CN100439499C - Neutrokine-alpha and Neutrokine-alpha splice variants - Google Patents

Abstract

The present invention relates to a novel cytokine known as Neutrokine-alpha. In addition, a splice variant of Neutrokine- α, designated Neutrokine- α SV, has been identified. In particular embodiments, the invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alpha SV polypeptides. In additional embodiments, Neutrokine-alpha and Neutrokine-alpha SV polypeptides are also provided, as well as vectors, host cells, and recombinant methods for producing them and uses thereof.

Description

Neutrokine-α and Neutrokine-α splice variants

The present invention relates to a novel cytokine which has been called Neutrokine-alpha. Additionally, an apparent splice variant of Neutrokine-α has been identified, termed Neutrokine-αSV. In specific embodiments, the invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alpha SV polypeptides. In additional embodiments, Neutrokine-alpha and Neutrokine-alpha SV polypeptides are also provided, as well as vectors, host cells, and recombinant methods of producing them.

related technology

Human tumor necrosis factors (TNF-α) and (TNF-β, or lymphotoxins) are related members of a large class of polypeptide mediators that includes interferons, interleukins, and growth factors, collectively referred to as cytokines (Beutler , B. and Cerami, A. Analytical Research in Immunology 7:625-655 (1989)). Sequence analysis of cytokine receptors has elucidated several subfamilies of membrane proteins: (1) Ig superfamily, (2) hematopoietin (cytokine receptor superfamily), and (3) tumor necrosis factor (TNF/ Nerve growth factor (NGF) receptor superfamily (with reference to TNF superfamily, found in Gruss and Dower, Blood 85(12):3378-3404 (1995) and Aggarwal and Natarajan, Eur. Cytokine Netw. 7(2):93- 124 (1996)). The TNF/NGF receptor superfamily contains at least 10 different proteins (Gruss and Dower, supra). The ligands for these receptors have been identified as belonging to at least two cytokine superfamilies (Gruss and Dower, as before).

Tumor necrosis factors (a mixture of TNF-α and TNF-β) were originally discovered for their antitumor activity, however they are now recognized as pleiotropic cytokines with diverse biological activities, including the ability to transform some cell lines Programmed death, mediates cell activation and proliferation, and also plays an important role in immune regulation and inflammation.

Known members of the TNF ligand superfamily include TNF-α, TNF-β (lymphotoxin-α), LT-β, OX40L, Fas ligand, CD30L, CD27L, CD40L, and 4-IBBL. Ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% (12%-36%) sequence homology in the extracellular domain and predominantly exhibit trimeric/multimeric complexes. Membrane-bound form of biologically active form. Soluble forms of the TNF ligand superfamily are currently only identified as TNF, β-LT, and Fas ligands (see Gruss, H and Dower, S.k., Blood 85 (12): 3378-3404 (1995), for all of them Incorporated by reference. These proteins are involved in the regulation of cell proliferation, activation and differentiation, including the control of cell survival or death by apoptosis or cytotoxicity (Armstage, R.J., Curr. Opin. Immund.b: 407 (1994) and Smith, C.A., Cell 75:959 (1994).

Tumor necrosis factor-α (TNF-α, also known as cachectin; hereinafter referred to as "TNF") is initially secreted by monocytes and macrophages in response to exotoxins or other stimuli, and is a A homotrimer of soluble 17KD protein subunit (Smith, R.A. et al., Biochemical Journal 262:6951-6954 (1987)). A membrane-bound 26KD precursor form of TNF has also been described (kriegIer , M. et al., Cell 53:45-53 (1988)).

Accumulated findings indicate that TNF is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipase synthesis ("cachesin" activity (Beutler, B. et al., Nature 316:552 (1985), activation of polymorphonuclear leukocytes (Klebanoff, S.J. et al., J. Immunol. 136:4220 (1986); Perussia, B. et al., J. Immunol. 138:765 (1987)), inhibiting or stimulating cell growth (Vilcek, J. et al., J. Exp. Med. 163:632 (1986); Sugarman, B.J. et al., Science 230:943 (1985); Lachman.L.B. et al., J. Immunol. 138:2913 (1987)), cytotoxic effects on certain transformed cells (Laehman, L.B. et al., supra; Darzynkiewicz, Z. et al., Cancer Research 44:83 (1984), antiviral activity (Kohase, M. et al., Cell 45:659 (1986); Wong, G.H.W. et al., Nature 323:819 (1986), stimulation of bone resorption (Bertolini, D.R. et al., Nature 319:516 (1986); Saklatvala, J., Nature 322:547 (1986), stimulates collagenase and prostaglandin F2 production (Dayer, J.-M. et al., J.Exp.Med.162:2163 (1985); and immunomodulatory activities, including activation of T cells (Yokota, S. et al., J. Immunology 140:531 (1988), activation of B cells (Kehrl.J.H. et al., J.Exp.Med. )), activate monocytes (Philip, R. et al., Nature 323: 86 (1986), activate thymocytes (Ranges, G.E. et al., J. Exp. Med. 167: 1472 (1988)), and stimulate major tissue phase Capacitance complex (MHC) class I and class II molecules are expressed on the cell surface (Couins, T. et al., Proc. USA 83:446 (1986); Pujol-Borrel, R. et al., Nature 326:304 (1987) ).

Note that TNF has a pro-inflammatory effect, leading to tissue damage, such as inducing procoagulation on vascular endothelial cells (Pober, J.S. et al., Journal of Immunology 136:1680 (1986), increasing the adhesion of neutrophils and lymphocytes (Pober, J.S. et al., J. Immunology 138:3319 (1987)), and stimulate the release of platelet-activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J.Exp.Med.166; 1390 (1987). Recent findings are about the role of TNF in many infections (Cerami.A et al., Current Immunology 9: 28 (1988)), immune disorders, tumorigenic pathologies, such as dyscrasias associated with some malignancies (Oliff , A et al., Cell 50:555 (1987)), and its pathological role in autoimmune pathology and graft-versus-host pathology (Piguet, P.F. et al., J Exp.Med.166:1280 (1987)). TNF The relationship to cancer and infection pathology often involves the catabolic state of the host. A major problem in cancer patients is weight loss, generally associated with anorexia. The result of continued consumption leads to what is known as "cachexia" (Kern, K.A. et al., J .Parent.Enter.Nutr.12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and malignant tumor growth leading to persistent erosion of constitution. Cachexia states are thus associated with overt morbidity and are associated with most cancer mortality Relatedly, many studies have speculated that TNF is an important mediator of cachexia in infectious pathology of cancer and in other catabolic states.

TNF is thought to play a role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H.R. et al., Br.J.Surg. 76:670-671 (1989); Debets, J.M.H et al., Second Vienna Shock Forum, P 463-466 (1989); Simpson, S.Q. et al. Crit.Care.Clin 5: 27-47 (1989), including fever, malaise, anorexia and dysphagia. Endotoxin is a potential monocyte/ Macrophage activator, stimulates the production and secretion of TNF (Kornbluth, S.K. et al., Journal of Immunology 137: 2585-2591 (1986)) and other cytokines. Since TNF can simulate the biological activity of many endotoxins, it is considered to be related to Major mediators associated with clinical manifestations of endotoxin-associated diseases. TNF and other monocyte-derived cytokines mediate metabolic and neurohormonal responses to endotoxin (Micdhie, H.R. et al., N.Eng.J.Med. 318:1481-1486 (1988). Administration of endotoxin to human volunteers produced acute illness with influenza-like symptoms including fever, tachycardia, increased metabolic rate and release of stress hormones (Revhauy, A. et al., Arch.Surg.123: 162-170 (1988)). It has also been found that circulating TNF levels are increased in patients with Gram-negative sepsis (Waage, A. et al., Lancetl: 355-357 ( 1987)); Hammerle, A.F et al., Second Vienna Shock Forum p.715-718 (1989); Debets, J.M.H. et al. Crit.Core.Med.17:489-497 (1989); Colandra T. et al., J.Infec. Dis.161:982-987(1990)).

Passive immunotherapy aimed at neutralizing TNF levels has a satisfactory effect on Gram-negative sepsis and endotoxemia, as mentioned above, based on increased TNF production and increased TNF levels in these pathological states. Cerami et al. (EPO Patent Publication 0212 489, March 4, 1987) disclose antibodies against a "modulator" identified as cachectin (later found to be identical to TNF). This antibody is useful in diagnostic immunoassays and in the treatment of shock in bacterial infections. Rubrn et al. (EPO patent publication 0218 868, April 22, 1987) disclosed a monoclonal antibody to human TNF, a hybridoma secreting this antibody, a method for producing this antibody, and this antibody in an immunoassay for TNF usage of. Yone et al. (Epo Patent Publication 0288088, October 26, 1988) disclose anti-TNF antibodies, including mAbs, and their role in immunoassay diagnosis of pathology, especially Kawasaki's pathology and cellular infection. TNF contained in body fluids of patients with Kawasakis pathology (acute febrile mucosal and cutaneous lymph node syndrome in children; Kawasaki, T., Auergg 16:178 (1967); Kawasaki, T., Shonica (Pediatrics) 26:935 (1985) Increased levels are associated with pathological processes (Yone et al., supra).

Other researchers have described mAbs specific to recombinant human TNF, which have neutralizing activity in vitro (Liang, C-M., etc., Biochemical Biophysiology Research Review 137:847-854 (1986); Medger, A., etc., Hybridoma 6: 305-311 (1987); Hirai, M. et al., J. Immunol. Methods 96: 57-62 (1987); Mouer, A. et al., Cytokine 2: 162-169 (1990)). Some of these mAbs were used for epitope mapping of human TNF and development of enzyme immunoassays (Fendly et al., supra; Hirai et al., supra; Mouer et al., supra), and to aid in the purification of recombinant TNF (Bringman et al., supra). As before) However, these studies cannot provide a basis for the production of TNF neutralizing antibodies that are diagnostic or therapeutic in vivo because of immunogenicity, lack of specificity and/or drug suitability.

Neutralizing antisera or mAbs to TNF have been shown to abolish adverse physiological changes and prevent death following experimental lethal studies in endotoxemia and bacteremia in mammals other than man. This effect has been demonstrated, for example, in rodent lethality assays and in primate pathological model systems (Mathison, J.C. et al. J. Clin. Investig. 81: 1925-1937 (1988); Beutler, B. et al., Science 229: 869-871 (1985) ; Tracey, K.J. et al., Nature 330:662-664 (1987); Shimamoto, Y. et al., Immunology Communications 17:311-318 (1988); Silva, A.T. et al., J. Infectious Diseases 162:421-427 (1990 ); Opal, S.M. et al., J. Infectious Diseases 161: 1148-1152 (1990); Hinshaw, L.B. et al., Circ. Shock 30: 279-292 (1990)).

It should be noted that trials of treatment with anti-TNF mAbs in humans are limited but have shown satisfactory therapeutic results, e.g. in the treatment of arthritis and sepsis. See for example Elliott, M.J. et al., Baillieres Clin. Rheumotol 9:633 -52 (1995); Feldmann M. et al., Ann.N.Y.Acad.Sci.USA 766:272-8 (1995); Poll, T. et al., Shock 3:1-12 (1995); Wheny et al., Crit.Care Med. 21: s436-40 (1993); Tyacey, K. J. et al., Crit. Care. Med. 21: S 415-22 (1993).

Mammalian development is dependent on cell proliferation and differentiation, and programmed cell death through apoptosis L. Walker et al., Methods Achiev. Exp. Pathol. 13:18 (1988). Apoptosis plays a key role in the destruction of immune thymocytes that recognize self-antigens. Disruption of this normal elimination process can lead to autoimmune disease (Gammon et al., Current Immunology 12:193 (1991)).

Itoh et al. (Cell 66:233 (1991)) describe a cell surface antigen, Fas/CD95, which mediates apoptosis and is involved in T cell clonal deletion. Fas is expressed in activated T cells, B cells, neutrophils, and in addition to activated T cells, B cells and neutrophils in the thymus, liver, heart and lung and ovary of adult mice Express. In experiments in which monoclonal Ab was cross-linked with Fas, apoptosis was induced (Yonehare et al., J. Experimental Methods 169:1747 (1989); Trauth et al., Science 245:301 (1989)). An example of Ab binding to Fas stimulating T cells under certain conditions (Alderson et al., J. Experimental Methods 178:2231 (1993)).

The Fas antigen is a cell surface protein of relevant MW of 45Kd. The human and mouse Fas genes have been cloned by Watanabe-Fukunaga et al. (J. Immunol. 148:1274 (1992)) and Itoh et al. (Cell 66:233 (1991)). The proteins encoded by these genes are all transmembrane proteins with structural homology to the nerve growth factor/tumor necrosis factor receptor superfamily, which includes two TNF receptors, the low-affinity nerve growth factor receptor and CD40, CD27, CD30 and OX40.

The Fas ligand has been described recently (Suda et al., Cell 75:1169 (1993)). The amino acid sequence indicated that Fas ligand is a type II transmembrane protein belonging to the TNF family. Thus, the FAS ligand polypeptide comprises 3 main domains: a short intracellular domain at the amino-terminus, a longer extracellular domain at the carboxyl-terminus, and a hydrophilic transmembrane domain linking the two domains. Fas ligand is expressed in splenocytes and thymocytes, consistent with T cell-mediated cytotoxicity. Purified Fas ligand has a MW of 40Kd.

In recent years it has been demonstrated that the interaction between Fas/Fas ligand is required for apoptosis following T cell activation (Ju et al., Nature 373:444 (1995); Brunner et al., Nature 373:441 (1995) ). Activation of T cells induces two proteins on the cell surface. Subsequent interactions between the ligand and receptor lead to programmed cell death. This supports a possible regulatory role for apoptosis induced by Fas/Fas ligand interactions during normal immune responses.

Therefore, there is a need to provide cytokines similar to TNF involved in pathological conditions. These new cytokines can be used to produce new antibodies or other antagonists combined with these TNF-like cytokines, so as to diagnose and treat diseases related to TNF-like cytokines.

Summary of the invention

According to one embodiment of the present invention, there is provided a novel extracellular domain of Neutrokine-α polypeptide, and a novel extracellular domain of Neutrokine-αSV polypeptide, and fragments thereof that are biologically active and used for diagnosis or treatment, similar to substances and derivatives.

According to another embodiment of the present invention, there are provided isolated nucleic acid molecules encoding human Neutrokine-α or Neutrokine-αSV, including mRNA, DNA, cDNA, genomic DNA, and fragments thereof that are biologically active and used for diagnosis or treatment and derivative.

The isolated nucleic acid molecules provided by the present invention comprise or consist of a polynucleotide encoding cytokines similar in structure to TNF and related cytokines, and having similar biological effects and activities and their apparent splice mutations body. This cytokine is called Neutrokine-α, and the Neutrokine-α polypeptide included in the present invention has at least a part of the amino acid sequence shown in Figure 1A and 1B (SEQ ID NO: 2), or has at least a part of it deposited on October 22, 1996 Amino acid sequence encoded by cDNA clone (HNEDU15) with deposit number 97768 in ATCC. The nucleotide sequence determined by sequencing the deposited Neutrokine-α clone is shown in Figures 1A and 1B (SEQ ID NO: 1), which contains an open reading frame encoding a complete polypeptide of 285 amino acid residues. Includes an N-terminal methionine, a deduced intracellular domain of approximately 46 amino acid residues, a deduced transmembrane domain of approximately 26 amino acid residues, a deduced extracellular domain of approximately 213 amino acids, and deduces the complete protein The molecular weight of about 31KDa. Like other type II transmembrane proteins, soluble forms of Neutrokine-α include all or part of the extracellular domain cleaved from the transmembrane domain, as well as polypeptides comprising the complete Neutrokine-α polypeptide lacking the transmembrane domain, i.e. linked to the intracellular domain extracellular domain.

The apparent splice variant of Neutrokine-α is called Neutrokine-αSV, and the Neutrokine-αSV included in the present invention comprises or at least a part of the amino acid sequence shown in Figure 5A and 5B (SEQ ID NO: 19), or by 1998 12 At least a part of the amino acid sequence encoded by the cDNA clone HDPMC52 deposited in ATCC with the accession number 203518 on March 10. The nucleotide sequence determined by sequencing the deposited Neutrokine-αSV clone is shown in Figures 5A and 5B (SEQ ID NO: 18), which contains an open reading frame encoding a complete polypeptide of 266 amino acids, including N-terminal methionine, a putative intracellular domain of about 46 amino acids, a putative transmembrane domain of about 26 amino acids, a putative extracellular domain of about 194 amino acids, and a deduced molecular weight of about 29 KDa for the complete protein. Like other type II transmembrane proteins, the soluble form of Neutrokine-αSV includes all or a portion of the extracellular domain cleaved from the transmembrane domain, and a polypeptide comprising the complete Neutrokine-αSV polypeptide lacking the transmembrane domain, i.e. linked to the intracellular domain extracellular domain.

Therefore one embodiment of the present invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide encoding a full-length Neutrokine-alpha polypeptide Sequence, the polypeptide has (SEQ ID NO: 2) shown in Figure 1A and 1B or is encoded by the cDNA clone contained in the deposit of ATCC deposit number 97768; An acid sequence, the ectodomain has the 73-285 amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2), or is encoded by a cDNA clone contained in the deposit of ATCC Accession No. 97768;

(c) a nucleotide sequence encoding a fragment of (b) polypeptide having Neutrokine-α functional activity (i.e. biological activity); (d) a nucleotide sequence encoding a polypeptide comprising a Neutrokine-α intracellular domain (presumed to be approximately 1-46 consecutive amino acid residues of Figures 1A and 1B (SEQ ID NO: 2)), or encoded by a cDNA clone contained in the deposit of ATCC Accession No. 97768; (e) encoding a polypeptide A nucleotide sequence comprising a Neutrokine-alpha transmembrane domain (presumed to be approximately 47-72 contiguous amino acid residues of Figures 1A and 1B (SEQ ID NO: 2)), or obtained from a deposit of ATCC Accession No. 97768 (f) encodes a nucleotide sequence encoding a soluble Neutrokine-α polypeptide having an extracellular domain and an intracellular domain but lacking a transmembrane domain; and (g) complementary to (a), (b), ( The nucleotide sequence of any of the nucleotide sequences in c), (d), (e) or (f).

Other embodiments of the present invention include isolated nucleic acid molecules comprising or consisting of polynucleotides having the same properties as (a), (b), (c), (d), (e) (f) above or any nucleotide sequence in (g) having at least 80%, 85% or 90% identity, preferably at least 95%, 96%, 97%, 98% or 99% identity, or the multinuclear The nucleotide hybridizes to the polynucleotide in (a), (b), (c), (d), (e) (f) or (g) above under stringent hybridization conditions. A polynucleotide to which it hybridizes does not hybridize under stringent conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or only T residues.

Another embodiment of the present invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide encoding a full-length Neutrokine-αSV polypeptide Sequence, the polypeptide has the complete amino acid sequence shown in Figures 5A and 5B (SEQID NO: 19), or has the cDNA contained in the deposit of ATCC deposit number 203518 deposited on December 10, 1998, cloned and encoded; (b ) encodes the nucleotide sequence of the putative extracellular domain of the Neutrokine-αSV polypeptide having the amino acid sequence 73-266 shown in Figures 1A and 1B (SEQ ID NO: 2) or deposited on December 10, 1998 The cDNA clone contained in the deposit of ATCC Accession No. 203518 encodes; (c) encodes a Neutrokine-αSV intracellular domain (presumed to be approximately 1-46 consecutive amino acid residues of Figures 5A and 5B (SEQ ID NO: 19)) The polynucleotide sequence of the polypeptide, the intracellular domain or the cDNA clone encoding contained in the deposit of ATCC deposit No. 203518 deposited on December 10, 1998; (d) encoding includes Neutrokine-αSV transmembrane domain (presumed to be 5A and 5B (SEQ ID NO: 19) the polynucleotide sequence of the polypeptide of about 47-72 contiguous amino acid residues), the transmembrane domain or from the deposit of ATCC deposit No. 203518 deposited on December 10, 1998 The cDNA clone coding containing; (e) the nucleotide sequence of encoding soluble Neutrokine-αSV polypeptide, this polypeptide has extracellular domain and intracellular domain but no transmembrane domain; And (f) is complementary to above (a), (b) , (c), (d), or the nucleotide sequence of any of the nucleotide sequences in (e).

Other embodiments of the present invention include isolated nucleic acid molecules comprising or consisting of polynucleotides having the same properties as (a), (b), (c), (d), (e) or (f) above. ) in any nucleotide sequence having at least 80%, 85% or 90% identity, preferably at least 95%, 96%, 97%, 98% or 99% identity of the nucleotide sequence, or the polynucleotide in strict hybridizes to the polynucleotide in (a), (b), (c), (d), (e) or (f) above under hybridization conditions. A polynucleotide to which it hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or only T residues.

In one embodiment, the apparent splice variant of Neutrokine-alpha comprises or consists of the sequence Gly142-Leu266 amino acids shown in Figures 5A and 5B (SEQ ID NO: 19), or deposited with the ATCC on December 10, 1998 It consists of at least a part of the amino acid sequence encoded by the cDNA clone HDPMC52 with the deposit number 203518.

In other embodiments, the nucleic acid molecule of the present invention comprises or consists of a polynucleotide encoding a polynucleotide having the above (a), (b), (c), (d), (e) (f) or ( Amino acid sequence of the epitope-carrying portion of Neutrokine-α or Neutrokine-αSV of the amino acid sequence in g). Another nucleic acid embodiment of the present invention relates to an isolated nucleic acid molecule comprising or consisting of a polynucleotide encoding the amino acid sequence of a Neutrokine-α or Neutrokine-αSV polypeptide having at least one But no more than 50, preferably no more than 40, more preferably no more than 30, most preferably no more than 20 amino acid additions, substitutions and/or deletions in the amino acid sequence. Of course, it is particularly preferred that the amino acid sequence of the polynucleotide encoding the amino acid sequence of Neutrokine-α or Neutrokine-αSV polypeptide contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or 1 - 100, 1-50, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acid additions, substitutions and/or deletions, preferably conservative substitutions.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, methods of producing such vectors and host cells, and methods of using them to produce Neutrokine-alpha polypeptides by recombinant techniques.

According to another embodiment of the present invention, there is provided a method for producing such a polypeptide by recombinant technology, comprising cultivating a recombinant prokaryotic containing the Neutrokine-α or Neutrokine-αSV nucleic acid sequence of the present invention under conditions that promote the expression of the polypeptide and/or eukaryotic host cells, followed by recovery of the polypeptide.

The present invention also provides an isolated Neutrokine-α polypeptide, which comprises or consists of an amino acid sequence selected from the group consisting of: (a) having the complete amino acid sequence shown in Figures 1A and 1B (i.e. 1-285 of SEQ ID NO: 2 position), or the amino acid sequence of the full-length Neutrokine-α polypeptide encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768; (b) has the complete amino acid sequence shown in SEQ ID NO: 2 except the N-terminal methionine (i.e. the 2-285 amino acid sequence of SEQ ID NO: 2) the amino acid sequence of the full-length Neutrokine-α polypeptide; (c) a fragment of (b) polypeptide with Neutrokine-α functional activity (i.e. biological activity); (d ) the amino acid sequence of the deduced extracellular domain of a Neutrokine-alpha polypeptide having the amino acid sequence 73-285 shown in Figures 1A and 1B (SEQ ID NO: 2), or the cDNA contained in the deposit of ATCC Accession No. 97768 Cloning code; (e) nucleotide sequence encoding Neutrokine-α polypeptide, having amino acid sequence 134-285 shown in Figure 1A and 1B (SEQ ID NO: 2); (f) amino acid of Neutrokine-α polypeptide intracellular domain sequence (approximately 1-46 contiguous amino acids of deduced Figures 1A and 1B (SEQ ID NO: 2)), the intracellular domain or encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768; (g) Neutrokine- Alpha transmembrane domain amino acid sequence (contiguous amino acid residues approximately 47-72 in deduced Figures 1A and 1B (SEQ ID NO: 2)), this transmembrane domain or the cDNA plasmid contained in the deposit of ATCC Accession No. 97768 (h) the amino acid sequence of a soluble Neutrokine-alpha polypeptide having an extracellular domain and an intracellular domain but no transmembrane domain, wherein said domains are as defined above; and (i), (a), (b), (c), (d), (e), (f), (g) or (h) a fragment of a polypeptide. The polypeptide of the present invention also includes at least 80% of the amino acid sequence of (a), (b), (c), (d), (e), (f), (g), (h) or (i) above, Preferably at least 85% or 90%, more preferably at least 95%, 96%, 97%, 98% or 99% amino acid sequence, and polypeptides having at least 80%, 85%, or 90% identity to the above amino acid sequences , more preferably polypeptides with amino acid sequences of at least 95% similarity. Another embodiment of the present invention relates to a polypeptide comprising or consisting of (a), (b), (c), (d), (e), (f), (g), (h) or ( Amino acid sequence composition of the epitope-carrying portion of the Neutrokine-α polypeptide of the amino acid sequence described in i). Neutrokine-alpha polypeptides of the present invention include such polypeptides having at least 4, at least 5, at least 6, at least 7, at least 8, preferably at least 9, at least 10, at least 11, at least 12, A portion of at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably at least about 30-50 amino acids, of course any length up to and including Polypeptides carrying epitopes of the entire amino acid sequence of the polypeptides of the present invention are also included in the present invention.

Particularly preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 80%, 85% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide , 90% identity, preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identity, Neutrokine-α polypeptide has 134- shown in Figure 1A and 1B (SEQ ID NO: 2) 285 amino acid sequence. Preferred embodiments of the invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence at least 90% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide, Neutrokine-alpha polypeptide It has the 134-285 amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2). More preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence at least 95% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide, Neutrokine-alpha The α-polypeptide has the 134-285 amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2). More preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence at least 96% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide, Neutrokine-alpha The α-polypeptide has the 134-285 amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2).

In addition, more preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence and a code having the 134- The polynucleotide sequence of the Neutrokine-α polypeptide at amino acid sequence 285 has at least 97% identity. In addition, more preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence and a code having the 134- The polynucleotide sequence of the Neutrokine-α polypeptide at amino acid sequence 285 has at least 98% identity. In addition, a more preferred embodiment relates to a nucleic acid component comprising or consisting of a polynucleotide having a nucleotide sequence and encoding having positions 134-285 shown in Figures 1A and 1B (SEQ ID NO: 2). The polynucleotide sequences of the Neutrokine-alpha polypeptides have at least 99% identity in amino acid sequence.

The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also encompassed by the present invention.

The present invention further provides an isolated Neutrokine-αSV polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of a full-length Neutrokine-αSV polypeptide having the complete amino acids shown in Figures 5A and 5B Sequence (i.e. amino acids 1-266 of SEQ ID NO: 19), or coded by a cDNA clone deposited in ATCC with accession number 203518 on December 10, 1998; (b) the amino acid sequence of the full-length Neutrokine-αSV polypeptide, The polypeptide has the complete amino acid sequence shown in SEQ ID NO: 19 except the N-terminal methionine (i.e. amino acids 2-266 of SEQ ID NO: 19); (c) the putative extracellular domain of Neutrokine-αSV polypeptide Amino acid sequence, the ectodomain has the 73-266 amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19), or is coded by a cDNA clone with a preservation number of 203518 deposited in ATCC on December 10, 1998; (d ) the amino acid sequence of the intracellular domain of the Neutrokine-αSV polypeptide, which is deduced to be approximately 1-46 consecutive amino acid residues shown in Figures 5A and 5B (SEQ ID NO: 19), or deposited on December 10, 1998 cDNA clone encoding at ATCC No.203518; (e) amino acid sequence of Neutrokine-αSV transmembrane domain, which is presumed to be approximately 47-72 consecutive amino acid residues shown in Figures 5A and 5B (SEQ ID NO: 19) or encoded by a cDNA clone deposited with ATCC No. 203518 on December 10, 1998; (f) the amino acid sequence of a soluble Neutrokine-αSV polypeptide having an extracellular domain and an intracellular domain but no transmembrane domain, wherein each each domain as defined above; and (g) a fragment of the above (a), (b), (c), (d), (e), or (f) polypeptide. The polypeptides of the present invention also include those having at least 80%, preferably at least 85% or 90% of the sequence described in (a), (b), (c), (d), (e), (f) or (g) above. %, more preferably at least 95%, 96%, 97%, 98% or 99% identical amino acid sequences, and polypeptides having at least 80%, 85% or 90% similarity, preferably at least 95% similarity to the aforementioned sequences Peptides with specific amino acid sequences. Another embodiment of the present invention relates to a polypeptide comprising or consisting of the amino acid sequence of the epitope-carrying portion of the Neutrokine-αSV polypeptide, the polypeptide having the above (a), (b), (c), (d), The amino acid sequence described in (e), (f) or (g). Peptides or polypeptides having the amino acid sequence of an epitope-carrying portion of a Neutrokine-αSV polypeptide of the present invention, including such polypeptides having at least 4, at least 5, at least 6, at least 7, at least 8, preferably at least 9 , at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably at least 30- A part of 50 amino acids, of course, any length up to and including the entire amino acid sequence of the polypeptide of the present invention, the epitope-carrying polypeptide is also included in the present invention.

Some non-limiting embodiments of the present invention relate to a polypeptide having the amino acid sequence of an epitope-carrying portion of a Neutrokine-α or Neutrokine-αSV polypeptide having (a) above, ( The amino acid sequence described in b), (c), (d), (e), (f), (g), (h) or (i). In other embodiments, the invention provides isolated antibodies that specifically (i.e. uniquely) bind to g), a Neutrokine-α or Neutrokine-αSV polypeptide of the amino acid sequence described in (h) or (i).

The present invention also provides a method for isolating an antibody that specifically (that is, uniquely) binds to a Neutrokine-α or Neutrokine-αSV polypeptide having the above-mentioned amino acid sequence. Such antibodies are useful in diagnosis or therapy as described below.

The present invention also provides a pharmaceutical composition comprising soluble Neutrokine-α and/or Neutrokine-αSV polypeptide, especially human Neutrokine-α and/or Neutrokine-αSV polypeptide, and/or anti-Neutrokine-α antibody and/or anti-Neutrokine-α Neutrokine-αSV antibodies, which can be used, for example, in the treatment, prevention, prognosis and/or diagnosis of tumors and tumor metastases, bacterial infections, viral and other parasitic infections, immunodeficiency, inflammation, lymph gland diseases, autoimmune diseases, grafts For host disease, stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation, survival and proliferation, mediate immune regulation and inflammatory responses, and enhance or suppress immune responses.

In some embodiments, soluble Neutrokine-α and/or Neutrokine-αSV polypeptides or agonists thereof of the present invention are administered to treat, prevent, prognose and/or diagnose immunodeficiency diseases (e.g., X-linked severe combined immunodeficiency (SCID) , autosomal SCID, adenosine deaminase deficiency (ADA deficiency), X-linked hypogammaglobulinemia (XLA), Bruton's disease, congenital hypogammaglobulinemia, X-linked hypogammaglobulinemia in children Hypoglobulinemia, Acquired Hypoglobulinemia, Adult Onset Hypoglobulinemia, Late Onset Hypoglobulinemia, Abnormal Hypoglobulinemia, Hypogammaglobulinemia, Pediatric transient hypogammaglobulinemia, non-specific hypogammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS) , X-linked hyper-IgM immunodeficiency, non-X-linked hyper-IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated levels of IgS, thymoma immunodeficiency, Ig weight Chain deletion, K chain deficiency, B-cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, occult hypogammaglobulinemia (Swiss type), reticulocyte hypoplasia, neonatal neutropenia syndrome, severe congenital leukopenia, thymic lymphoid hypoplasia-hypoplasia or dysplasia immunodeficiency, ataxia telangiectasia, short-limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezolf combined with IgS immunodeficiency syndrome , purine nucleoside phosphorylase (PNP) deficiency, MHC class II deficiency (Bare lymphocytic syndrome), and severe combined immunodeficiency), or disorders associated with immunodeficiency.

In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the invention, or agonists thereof, are administered for the treatment, prevention, prognosis and/or diagnosis of common variable immunodeficiency.

In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or their agonists, are administered for the treatment, prevention, prognosis and/or diagnosis of X-linked blood gamma globulin hypoxia.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or their agonists, are administered for the treatment, prevention, prognosis and/or diagnosis of severe combined immunodeficiency (SCID) ).

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the invention, or agonists thereof, are administered for the treatment, prevention, prognosis and/or diagnosis of Wiskott-Aldrich syndrome.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or their agonists, are administered for the treatment, prevention, prognosis and/or diagnosis of X-linked high IgM levels Ig deficiency.

In another embodiment, Neutrokine-α antagonists and/or Neutrokine-αSV antagonists (such as anti-Neutrokine-α antibodies) are administered for the treatment, prevention, prognosis and/or diagnosis of autoimmune diseases (such as rheumatoid arthritis, Systemic lupus erythematosus, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, renal Glomerulonephritis (eg, IgA nephropathy), multiple sclerosis, neuritis, uveitis eye disease, purpura (eg, Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroid disease, hypothyroidism (ie, Hashimoto's thyroid disease), Goodpasture's syndrome, pemphigus, recipient autoimmunity such as ( a) Grave's disease, (b) Myasthenia Grave, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, anti-collagen antibody schleroderma, connective tissue disease, polymyositis/dermatomyositis inflammation, pernicious anemia, primary Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, Siogren's syndrome, diabetes mellitus, and Epinephrine drug resistance (including adrenaline drug resistance in asthma or cystic fibrosis), chronic active hepatitis, primary gallbladder sclerosis, other endocrine gland disorders, vitiligo, vasculitis, post-MI, cardiotomy syndrome, rubella, atopic dermatitis, asthma, inflammatory myopathy, and other inflammatory, granulamatous, degeneration, and atrophy) or diseases associated with autoimmune diseases. In a specific preferred embodiment, anti-Neutrokine-α antibodies and/or anti-Neutrokine-αSV antibodies and/or other antagonists of the present invention are used to treat, prevent, prognose and/or diagnose rheumatoid arthritis. In another specific preferred embodiment, the treatment, prevention, prognosis and/or diagnosis of systemic lupus erythematosus. In another preferred embodiment, anti-Neutrokine-α antibody and/or anti-Neutrokine-αSV antibody and/or other antagonists are used for treatment, prevention, prognosis and/or diagnosis of idiopathic thrombocytopenic purpura. In another preferred embodiment, the anti-Neutrokine-α antibody and/or anti-Neutrokine-αSV antibody and/or other antagonists of the present invention are used for the treatment, prevention, prognosis and/or diagnosis of IgA nephropathy. In a preferred embodiment, anti-Neutrokine-α antibody and/or anti-Neutrokine-αSV antibody is used for treatment, prevention, prognosis and/or diagnosis of autoimmune diseases related to the above diseases and dysfunction, and dysfunction and/or lesion.

The present invention further provides a composition comprising Neutrokine-α or Neutrokine-αSV polynucleotide, Neutrokine-α or Neutrokine-αSV polypeptide, and/or anti-Neutrokine-α antibody or anti-Neutrokine-αSV antibody, administered in vitro A cell of, a cell from a body, a cell in a body, or a multicellular organism. In a preferred embodiment, the composition of the present invention comprises Neutrokine-α and/or Neutrokine-αSV polynucleotide, so as to express Neutrokine-α polypeptide and/or Neutrokine-αSV polypeptide in the host body, thereby treating diseases. In a more preferred embodiment, the composition of the present invention comprises Neutrokine-α and/or Neutrokine-αSV polynucleotide, so as to express Neutrokine-α and/or Neutrokine-αSV polypeptide in the host body, thereby treating immunodeficiency Disease or a disease associated with immunodeficiency. Particularly preferred is expression in a patient for the treatment of dysfunction associated with abnormal endogenous activity of the Neutrokine-α or Neutrokine-αSV gene, (e.g. enhanced expression of abnormal B cell function by increasing B cell numbers or increasing B cell longevity) .

The present invention also provides a screening method to identify compounds capable of enhancing or inhibiting the cellular response induced by Neutrokine-α and/or Neutrokine-αSV, the method comprising expressing Neutrokine-α and/or Neutrokine-αSV and The selected compound is contacted, the cellular response is analyzed, and compared with the standard cellular response, which is analyzed in the absence of the candidate compound; thus, the high cellular response and the standard response indicate that the compound is an agonist, and the cellular response is lower than that of the candidate compound. Standard responses indicate that the compound is an antagonist.

In another embodiment, a method of identifying Neutrokine-[alpha] and/or Neutrokine-[alpha]SV receptors, and methods of screening for agonists and antagonists using such receptors are provided. This analysis involves determining the effect of candidate compounds on the binding of Neutrokine-α and/or Neutrokine-αSV to the Neutrokine-α and/or Neutrokine-αSV receptor. In particular, the method comprises contacting Neutrokine-α and/or Neutrokine-αSV receptors with Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention and a candidate compound, and determining that due to the presence of the candidate compound, Neutrokine-α and/or Or whether the binding of Neutrokine-αSV polypeptide to Neutrokine-α and/or Neutrokine-αSV receptor is increased or decreased. Antagonists can be used to prevent septic shock, inflammation, cerebral malaria, HIV viral activation, graft/host rejection, bone resorption, rheumatoid arthritis, cachexia (wasting or dystrophic), immune system dysfunction, lymphoma, and autoimmune disorders (such as rheumatoid arthritis and systemic lupus erythematosus).

The present inventors also revealed that Neutrokine-α is expressed not only in monocytic cell lines, but also in kidney, lung, peripheral blood cells, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, bone cancer, smooth muscle, macrophage cells, and umbilical cord blood tissue. The present inventors further revealed that Neutrokine-αSV appears to be highly expressed only in primitive dendritic cells. Some disorders of these tissues and cells, such as tumors and tumor metastasis, bacterial, viral and other parasitic infections, immunodeficiency (eg, chronic variable immunodeficiency), septic shock, inflammation, brain abuse, HIV viral reactivation, graft/host Rejection, bone resorption, rheumatoid arthritis, autoimmune diseases (eg, rheumatoid arthritis, and systemic lupus erythematosus), and dyscrasias (wasting or dystrophic). It is believed that significantly higher or lower levels of Neutrokine-α and/or Neutrokine-[alpha]SV gene expression, "standard" Neutrokine-[alpha] or Neutrokine-[alpha]SV gene expression level is the expression level in tissues or body fluids taken from individuals without the disorder. Therefore, the present invention provides a diagnostic method used during the diagnosis of a disease, comprising (a) analyzing the expression level of Neutrokine-α and/or Neutrokine-αSV gene in individual cells or body fluids; (b) comparing the expression level with standard Neutrokine-αSV gene expression level; α and/or Neutrokine-αSV gene expression levels are compared, and the comparison results are higher or lower than the standard level to indicate the presence of the disease.

Other embodiments of the invention relate to a method of treating an individual in need of increasing or maintaining levels of Neutrokine-α and/or Neutrokine-αSV activity in vivo comprising administering to such an individual a composition comprising a therapeutically effective amount of Isolated Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention or agonists thereof.

Another embodiment of the present invention is directed to a method of treating an individual in need of reduced levels of Neutrokine-α and/or Neutrokine-αSV activity in the body comprising administering to such individual a composition comprising a therapeutically effective amount of Neutrokine - alpha and/or Neutrokine-alpha SV antagonists. Preferred antagonists for use in the present invention are Neutrokine-α-specific antibodies and/or Neutrokine-αSV-specific antibodies.

Brief description of the drawings

The following figures illustrate embodiments of the present invention without limiting the scope of the claims of the present invention.

Figures 1A and 1B show the nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2) sequences of Neutrokine-α. Amino acids 1-46 represent the putative intracellular domain, amino acids 47-72 represent the putative transmembrane domain (underlined), and amino acids 73-285 represent the putative extracellular domain (the rest of the sequence). Potential asparagine-linked glycosylation sites are indicated in Figures 1A and 1B by the bold asparagine symbol (N) in the Neutrokine-α amino acid sequence encoding the asparagine residue in the Neutrokine-α nucleotide sequence The first nucleotide of the base has a bold # sign. Potential N-linked glycosylation sequences were found at the following positions in the Neutrokine-α amino acid sequence: N124-Q127 (N-124, S-125, S-126, Q-127) and N242-C245 (N-242, N -243, S-244, C-245).

Regions of high identity between Neutrokine-α, Neutrokine-αSV, TNF-α, TNF-β, LT-β, and the closely related Fas ligand (Figure 2 shows the alignment of these sequences) are underlined in Figures 1A and 1B . These regions are not limiting and are labeled in Figures 1A and 1B as conserved domains (CD)-I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD -VIII, CD-IX, CD-X, and CD-XI.

Figures 2A and 2B show Neutrokine-α (SEQ ID NO: 2) and Neutrokine-α SV (SEQ ID NO: 19), and TNF-α ("TNF-α" in Figures 2A and 2B, GenBank No.Z15026; ( SEQ ID NO: 3), TNF-β ("TNF-β" in Figure 2A and 2B, GenBank No.Z15026; SEQ ID NO: 4), lymphotoxin-β ("LT-β" in Figure 2A and 2B, GenBank No.L11016; SEQ ID NO: 5), and the region of identity between the amino acid sequences of the FAS ligand ("FASL" in Figures 2A and 2B, GenBank No.U1182I; SEQ ID NO: 6), which was identified by the "MegAlign" program Established, this program is part of a computer program called "DAN*STAR". Residues that match the consensus sequence are shaded.

Figure 3 shows Neutrokine-α amino acid sequence analysis. The amino acid sequence of SEQ ID NO:2 is deduced with the default parameters of the computer program, showing α region, β region, turn region and coil region; hydrophilicity and hydrophobicity; amphipathic region; flexible region; antigenic index and surface probabilities. In the "antigen index-Jameson-Wolf' figure, the position of the highly antigenic region of Neutrokine-α is indicated, which is the region from which the epitope-carrying peptide of the present invention can be obtained. Antigenic polypeptides include SEQ ID NO: 2 About Phe115~Leu147, Ile150~Tyr163, Ser171~Phe194, Glu223~Tyr246, Ser271~Phe278.

The data in Figure 3 are also shown in Table 1 in tabular form. Columns are labeled with "residue", "position" and Roman numerals I-XIV. Column labels refer to the following features of the amino acid sequences shown in Figure 3 and Table 1: "residue" refers to the amino acid residue of SEQ ID NO: 2 and Figures 1A and 1B; "position" refers to SEQ ID NO: 2 and Figure 1A and the positions of corresponding residues within 1B; I: α region-Garnier-Robson; II: α region-Chou-Fasman; III: β region-Garnier-Robson; IV: β region-Chou-Fasman; V: turn region- Garnier-Robson; VI: corner region-Chou-Fasman; VII: coil region-Garnier-Robson; VIII: hydrophilicity map-Kyte-Doolittle; IX: hydrophobicity map-Hopp-Woods; X: α amphipathic region- Eisenberg; XI: Beta Amphiphilic Region - Eisenberg; XII: Flexible Region - Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Map - Emini.

Figures 4A, 4B and 4C show clones of human cDNAs related to the present invention, designated HSOAD55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8) and HLTBM08 (SEQ ID NO: 9), with reference to those deposited at Alignment of the Neutrokine-α nucleotide sequence determined from the human cDNA clone of ATCC No. 97768.

Figures 5A and 5B show the nucleotide sequence (SEQ ID NO: 18) and deduced amino acid sequence (SEQ ID NO: 19) of the Neutrokine-αSV protein. Amino acids 1-46 represent the putative intracellular domain, amino acids 47-72 represent the putative transmembrane domain (underlined double line), and amino acids 73-266 represent the putative extracellular domain (the rest of the sequence). Potential asparagine-linked glycosylation sites are marked in Figures 5A and 5B with the asparagine symbol in bold (N) in the Neutrokine-αSV amino acid sequence and asparagine in the nucleotide sequence encoding Neutrokine-αSV Residues are marked with a # in bold above the first nucleotide. Potential N-linked glycosylation sequences were found at the following positions in the Neutrokine-αSV amino acid sequence: N124~Q127 (N124, S125, S126, Q127), and N223~C226 (N223, N224, S225, C226). Antigenic polypeptides include about Pro32-Leu47, Glu116-Ser143, Phe153-Tyr173, Pro218-Tyr227, Ala232-Gln241, Ile244-Ala249, and Ser252-Val257 of the amino acid sequence of SEQ ID NO: 19.

Regions of high identity between Neutrokine-α, Neutrokine-αSV, TNF-α, TNF-β, LT-β, and the closely related Fas ligand (the alignment of these sequences is shown in Figure 2) are underlined in Figures 1A and 1B place. (of Neutrokine-α and Neutrokine-αSV) These conserved regions are marked as conserved domains (CD)-I, CD-II, CD-III, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI. Neutrokine-αSV does not contain the CD-IV sequence described in the legends of Figures 1A and 1B.

The sequence alignment of Neutrokine-α polypeptide sequence (SEQ ID NO: 2) with APRIL, TNF-α, LT-α is shown in Figure 7A. In Figure 7A, the beta sheet region is shown as described below in the Figure 7A legend.

Figure 6 shows the analysis of the Neutrokine-αSV amino acid sequence, using the default parameters of the computer program to deduce the alpha region of the amino acid sequence of SEQ ID NO: 19, the beta region, the corner region and the coil region; hydrophilicity and hydrophobicity; amphipathic area; flexible area; antigen index and surface probability. The position of the highly antigenic region of the Neutrokine-α protein, which is the region from which the epitope-bearing peptides of the present invention can be obtained, is shown in the "antigenicity index - Jameson-Wolf" diagram. Antigenic polypeptides include, but are not limited to, polypeptides comprising the following amino acids of the amino acid sequence of SEQ ID NO: 19: about Pro32-Leu47, about Glu116-Ser143, about Phe153-Tyr173, about Pro218-Tyr227, about Ser252-Thr258, about Ala232- Gln241, about Ile244-Ala249, about Ser252-Val257.

The data shown in Figure 6 can be represented in tabular form similar to the data shown in Table 1. Such a table representing the exact data shown in Figure 6 can be generated using the MegAligh component of the DNA*STAR computer sequence analysis package, with default parameters set up. This is the same procedure used to generate Figures 3 and 6.

Figure 7A: The amino acid sequence of Neutrokine-α, and the sequence alignment of its putative ligand-binding domain and the ligand-binding domain of APRIL, TNF-α, LT-α (specifically, human APRIL polypeptide (SEQ ID NO: 20; 115-250 amino acid residues of ATCC deposit No.AF046888), 88-233 amino acid residues of TNF-α (SEQ ID NO: 3; GenBank No. Z15026), and LT-α (also known as TNF-β , 62-205 amino acid residues of SEQ ID NO: 4); GenBankNo.Z15026). The putative transmembrane region of Neutrokine-α is shown and the cleavage site of Neutrokine-α is in italics. Underlined sequences (A-H) represent putative β-sheet regions.

Figure 7B: Neutrokine-α mRNA expression. Northern hybridization analysis was performed on poly(A) ⁺ Northern blots (Clonetech) obtained from a range of human tissue types and selected cancer cell lines using the Neutrokine-[alpha] open reading frame as a probe. A 2.6Kb Neutrokine-α mRNA was detected to be highly expressed in placenta, heart, lung, fetal liver, thymus and pancreas. This 2.6Kb Neutrokine-α mRNA was also detected in HL-60 and K562 cell lines.

Figures 8A and 8B: Increased expression of Neutrokine-α following activation of human monocytes by IFN-γ. Figure 8A: Flow cytometric analysis of Neutrokine-α protein expression on monocytes cultured in vitro. Purified cells were cultured for 3 days with or without IFN-γ (100 U/ml). Cells were then stained with Neutrokine-α-specific mAb (2E5) (solid line) or an isotype-matched control (IgG1) (dashed line). Comparable results were obtained with monocytes purified from three different donors in three independent experiments. Figure 8B: Neutrokine-α specific TagMan primers were prepared and used to determine the relative expression levels of Neutrokine-α mRNA in unstimulated and IFN-γ (100 U/ml) treated monocytes. The nucleotide sequences of TagMan primers are as follows: (a) probe: 5′-CCA CCA GCT CCA GGA GAA GGC AAC TC-3′ (SEQ ID NO: 24); (b) 5′ amplification primer: 5′- ACC GCG GGA CTG AAA ATC T-3' (SEQ ID NO: 25); and (c) 3' amplification primer: 5'-CAC GCT TAT TTC TGC TGTTCT GA-3' (SEQ ID NO: 26).

Figures 9A and 9B: Neutrokine-α is a potential B-lymphocyte stimulator. Figure 9A: Bioactivity of Neutrokine-α was determined in a standard B lymphocyte co-stimulation assay using Staphylococcus aureus cowan 1 (SAC) as a lead reagent. SAC alone produced a background count of 1427±316. Reported values are mean ± deviation of results obtained in triplicate wells. Similar results were obtained with recombinant Neutrokine-α purified from stable CHO transfectants and transiently transfected HEK293T cells. Figure 9B: Stimulation of proliferation of tonsillar B cells with Neutrokine-α and its combination with anti-IgM. Bioassays were performed as described for analysis with SAC, except that individual wells were pre-coated with goat anti-human IgM antibody at a concentration of 10 μg/ml PBS.

Figures 10A and 10B: Neutrokine-alpha receptor expression in normal human peripheral blood mononuclear cells and tumor cell lines. Figure 10A: Human peripheral blood nucleated cells were obtained from normal volunteers and isolated by density gradient centrifugation. Cells were stained with biotinylated Neutrokine-α followed by PE-conjugated streptavidin and FITC- or PerCP-conjugated mAbs specific for CD3, CD20, CD14, CD56 and CD66b. Cells were analyzed with CellQuest software on a Becton Dickinson FACScan. Data represent one of 4 independent experiments. Figure 10B: Binding of Neutrokine-α to the histiocyte cell line U-937 and the myeloma cell line IM-9.

Figures 11A, 11B, and 11C: In vivo effects of Neutrokine-α administration in BALB/cAn NCR mice. Figure 11A: Formalin-fixed spleens were embedded in paraffin and 5 micron sections were stained with hematoxylin and eosin (upper panel). The lower panel is a section taken from the same animal that was stained with anti-CD45R(B220) mAb and treated with horseradish peroxidase-conjugated rabbit anti-mouse Ig (mouse-adsorbed) and the substrate tetrahydrochloride diamino-linked Aniline (DAB) color development. Slides were negatively stained with Mayer's hematoxylin. CD45R(B220) expressing cells are brown. Figure 1 IB: Flow cytometric analysis of normal (left panel) and Neutrokine-α-treated (right panel) stained with PE-CD45R (B220) and FITC-ThB (LybD). Figure 11C: Serum IgM, IgG and IgA levels in normal and Neutrokine-α-treated mice.

A detailed description

The present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding a Neutrokine-alpha polypeptide having the amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2), which sequence is determined by sequencing cDNA clones of. The nucleotide sequence (SEQ ID NO: 1) shown in Figure 1A and 1B is obtained by sequencing the HNEDU15 clone, which was deposited in the American Type Culture Collection (ATCC) on October 22, 1996. ATCC No. 97768, located at 20110-2209 University Drive, Manassas, Virginia, 10801. The deposited clone was contained in the pBluescriptSK(-) plasmid (Stratagene, La Jolla, CA).

The present invention also provides an isolated nucleic acid molecule comprising a polynucleotide encoding a Neutrokine-αSV polypeptide having the amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19), which is obtained by sequencing cDNA clones definite. The nucleotide sequence (SEQ ID NO: 18) shown in Figure 5A and 5B is obtained by sequencing the HDPMC52 clone, which was deposited in the American Type Culture Collection (ATCC) on December 10, 1998. No. ATCC No. 203518. The deposited clone was contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).

Neutrokine-α and Neutrokine-αSV polypeptides of the present invention are sequence homologous to translation products of human mRNAs of TNF-α, TNF-β, LT-β, Fas ligand, APRIL and LT-α (see Fig. 2A, 2B and 7A). As indicated above, TNF-α is thought to play a role in cytotoxicity, necrosis, programmed cell death, costimulation, proliferation, lymphangiogenesis, immunoglobulin class switching, differentiation, antiviral activity, and regulation of adhesion molecules and other cytokines An important cytokine that plays a role in growth factors.

nucleic acid molecule

Unless otherwise specified, all nucleotide sequences determined by sequencing the DNA molecules herein were determined using an automatic DNA sequencer (such as Model 373, Applied Brosystems, Inc, Foster City, CA), and determined by the DNA All amino acid sequences of polypeptides encoded by the molecules were deduced by translation of the DNA sequences identified above. Accordingly, it is known in the art that any nucleotide sequence determined herein, like any DNA sequence determined by such automated methods, is subject to some error. The nucleotide sequence determined by automated methods is typically at least about 90%, more typically at least about 95% to 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be determined more precisely by other methods, including manual DNA sequencing methods well known in the art. It is also known in the art that a single insertion or deletion in a defined nucleotide sequence, compared to the true sequence, will result in a frameshift in translation of the nucleotides, whereby from the point of such insertion or deletion, The deduced amino acid sequence encoded by the determined nucleotide sequence will be completely different from the actual amino acid sequence encoded by the sequenced DNA molecule.

The "nucleotide sequence" of a nucleic acid molecule or polynucleotide is the deoxyribonucleotide sequence for a DNA molecule or polynucleotide and the corresponding ribonucleotide sequence for an RNA molecule or polynucleotide (A , G, C and U), wherein each thymidine (T) deoxyribonucleotide in the specific deoxyribonucleotide sequence is replaced by a ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequences shown in Figures 1A and 1B, nucleic acid molecules of the invention encoding Neutrokine-alpha polypeptides can be obtained using standard cloning and screening methods, such as cloning cDNA using mRNA as a starting material. As shown in the present invention, the nucleic acid molecule (SEQ ID NO: 1) described in Figures 1A and 1B was found in a cDNA library derived from neutrophils. An expressed sequence tag corresponding to a portion of Neutrokine-α cDNA is also found in kidney, lung, peripheral blood leukocytes, bone marrow, T-cell lymphoma, B-cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages, and cord blood tissues middle. Alternatively, using the nucleotide information provided in Figures 5A and 5B, nucleic acid molecules of the invention encoding Neutrokine-αSV polypeptides can be obtained using standard cloning and screening methods, such as cloning cDNA using mRNA as a starting material. As shown in the present invention, the nucleic acid molecule (SEQ ID NO: 18) shown in Figures 5A and 5B was found in a cDNA library derived from primitive dendritic cells.

The Neutrokine-alpha plasmid HNEDU15 of ATCC deposit number 97768 contains an open reading frame encoding a protein of about 285 amino acid residues, a putative intracellular domain of about 46 amino acids (Figures 1A and 1B (SEQ ID NO: 2) about amino acid residues 1-46 of ), a putative transmembrane domain of about 26 amino acids (underlined amino acid residues about 47-72 in Figures 1A and 1B (SEQ ID NO: 2), an about 213 putative ectodomain of amino acids (approximately 73-285 amino acids in Figures 1A and 1B (SEQ ID NO: 2)); the deduced molecular weight is approximately 31 KDa. The Neutrokine-α polypeptide shown in Figures (1A and 1B (SEQ ID NO: 2) has about 20% similarity and about 10% identity to human TNF-α, which has accession number No.339764 in GenBank.

The Neutrokine-αSV plasmid HDPMC52 of ATCC deposit number 203518 contains an open reading frame encoding a protein of about 266 amino acid residues, a putative intracellular domain of about 46 amino acids (Figure 5A and 5B (SEQ ID NO: 19) about 1-46 amino acid residues), a putative transmembrane domain of about 26 amino acids (underlined about 47-72 amino acid residues in Figures 5A and 5B (SEQ ID NO: 19), an about 194 Putative ectodomain of amino acids (approximately amino acid residues 73-266 in Figures 5A and 5B (SEQ ID NO: 19)); deduced molecular weight of approximately 29 KDa. The Neutrokine-αSV polypeptide shown in Figures 5A and 5B (SEQ ID NO: 19) has approximately 33.9% similarity and approximately 22.0% identity to human TNF-α with GenBank accession number 339764.

The skilled artisan is aware that the actual complete Neutrokine-α and/or Neutrokine-αSV polypeptides encoded by the deposited cDNAs, comprising approximately 285 and 266 amino acids, respectively, may sometimes be somewhat shorter due to possible sequencing errors as described above. In particular, the identified Neutrokine-α and Neutrokine-αSV coding sequences contain a common second methionine codon, which serves as an initiation codon for translation of the open reading frame, which is located in Figures 1A and 1B ( 64-66 nucleotides shown in SEQ ID NO: 18). More generally, the true open reading frame can be deduced from the N-terminal first or second methionine shown in Figures 1A and 1B (SEQ ID NO: 1) and Figures 5A and 5B (SEQ ID NO: 18). Any position within ±20 amino acids, especially ±10 amino acids, of the codon. It should further be appreciated that the polypeptide domains described herein are determined by computer analysis, and thus, the extracellular, intracellular, and transmembrane domains of Neutrokine-α and Neutrokine-αSV polypeptides are determined according to the analytical criteria used to identify the various domains. The exact location can vary. For example, the exact positions of the Neutrokine-α and Neutrokine-αSV ectodomains in Figures 1A and 1B (SEQ ID NO: 2) and Figures 5A and 5B (SEQ ID NO: 19) may vary slightly depending on the criteria used to define the domain (e.g. , the position can be "shifted" by about 1-20 residues, especially about 1-5 residues). In this case, the terminus of the transmembrane domain and the start of the extracellular domain were deduced based on the identification of the hydrophobic amino acid sequences in the positions indicated above, as shown in Figures 3 and 6 and Table 1. As described below, the invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of the complete polypeptide, including polypeptides lacking one or more of the N-terminal amino acids of the extracellular domain described herein, which constitute Soluble forms of Neutrokine-α and the extracellular domain of Neutrokine-αSV polypeptides.

The nucleic acid molecules and polynucleotides of the invention may be in the form of RNA, such as mRNA, or DNA, including cDNA and genomic DNA obtained by cloning or synthetically produced. This DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also known as the antisense strand.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that is separated from its natural environment. For example, a recombinant DNA molecule contained in a vector of the invention is considered isolated. Isolated DNA molecules also include, for example, recombinant DNA molecules maintained in heterologous host cells, or purified (partially or substantially purified) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules of the invention. However, nucleic acids contained in clones, or chromosomes isolated or removed from cells or cell lysates (e.g., karyotyped "chromosomal smears") are not isolated in the present invention, and the clones are not separated from other members of the library ( A member of a library (eg, a genomic or cDNA library) isolated such as in the form of a homogeneous solution containing the clone and other library members. As further described herein, an isolated nucleic acid molecule according to the invention may be naturally, recombinantly or synthetically produced.

The isolated nucleic acid molecule of the present invention includes a DNA molecule comprising or consisting of an open reading frame (ORF) with an initiation codon at positions 147-149 of the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1). )composition. In addition, the DNA molecule included in the isolated nucleic acid molecule of the present invention comprises or consists of a sequence that is substantially different from the above-mentioned sequence, but still encodes Neutrokine-α protein due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Thus, degenerate variants of the above can be routinely generated by those skilled in the art. In another embodiment, the isolated nucleic acid molecule provided herein comprises or consists of a sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence encoded by the cDNA contained in the plasmid with ATCC Accession No. 97768. Preferably, the nucleic acid molecule comprises or consists of a mature or soluble polypeptide sequence encoding the extracellular domain of a polypeptide encoded by the cDNA in the plasmid of ATCC Deposit No. 97768.

The isolated nucleic acid molecule of the present invention also includes a DNA molecule comprising an open reading frame (ORF ). In addition, the DNA molecules included in the isolated nucleic acid molecules of the present invention comprise or consist of sequences that are substantially different from the sequences described above, but still encode Neutrokine-αSV polypeptides due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Thus, the degenerate variants described above can be routinely produced by those skilled in the art. In another embodiment, the isolated nucleic acid molecule provided herein comprises or consists of a sequence encoding a Neutrokine-αSV polypeptide having amino acids encoded by the cDNA contained in the plasmid with ATCC Accession No. 203518. Preferably, the nucleic acid molecule comprises or consists of a sequence encoding the ectodomain or mature soluble polypeptide sequence of a polypeptide encoded by the cDNA contained in the plasmid of ATCC Deposit No. 203518.

The nucleic acid molecule further provided by the present invention comprises or is by the nucleotide sequence shown in Fig. 1A and 1B (SEQ IDNO: 1), or is contained in the nucleotide sequence of the Neutrokine-α cDNA in the plasmid of ATCC No.97768, or has complementary The nucleotide sequence composition of one of the above sequences. In addition, the isolated nucleic acid molecule provided by the invention comprises or is represented by the nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO: 18), or the nucleotides of the Neutrokine-αSV cDNA contained in the plasmid of ATCC No.203518 sequence, or a nucleotide sequence having a sequence complementary to one of the above sequences. Such isolated molecules, especially DNA molecules, have useful properties including, but not limited to, as probes for gene mapping by in situ hybridization to chromosomes, and for the detection of Neutrokine-α and Neutrokine-αSV in human tissues, for example by Northern or Western blot analysis. expression etc.

In one embodiment, the polynucleotide of the present invention comprises or consists of the sequence shown in SEQ ID NO:22. The sequence shown in SEQ ID NO: 22 was constructed from several overlapping mouse EST sequences (AI 182472, AA 422749, AA 25047 and AI 122485) obtained from GenBank. The EST sequences were compared to generate the Neutrokine-alpha polynucleotide sequence shown in SEQ ID NO: 22. The amino acid sequence derived from the translation of SEQ ID NO:22 is shown in SEQ ID NO:23. Fragments, variants and derivatives of the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23 are also encompassed by the present invention.

In another embodiment, the polynucleotide of the present invention comprises or consists of the sequence shown in SEQ ID NO: 27, and/or the sequence encoding the amino acid sequence shown in SEQ ID NO: 28, fragments, variants and derivatives thereof . These polynucleotides are also encompassed by the present invention. For example, some embodiments of the present invention relate to polynucleotides comprising or consisting of a sequence encoding a polypeptide sequence, which has at least 80%, 85%, or 90% of amino acids 68-219 of SEQ ID NO: 28 , 92%, 95%, 96%, 97%, 98% or 99% identity. The amino acid sequence derived from the translation of SEQ ID NO:27 is shown in SEQ ID NO:28. Polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 28 and fragments, variants and derivatives of the sequence shown in SEQ ID NO: 28 are also encompassed by the present invention. For example, some embodiments of the present invention relate to polypeptides comprising or having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% of amino acids 68-219 of SEQ ID NO: 28 % or 99% identical polypeptide sequence composition. The nucleic acid molecule having the sequence shown in SEQ ID NO: 27 is obtained from cyanomoloous monkey (i.e. Macaea irus) PBMC by RT-PCR through two kinds of degenerate primers. Briefly, total RNA was prepared from cyanomologous monkey PBMCs by using Trizol (purchased from Life Technologies, Inc, Rockville, MD) according to the manufacturer's instructions. Single-stranded cDNA was then synthesized from cyanomoloous monkey PBMC preparations using oligo-dT primers using standard methods. Neutrokine-α-specific primers were designed based on the conserved region between mouse and human Neutrokine-α molecules (SEQ ID NO: 22 and 1, respectively). The following two degenerate oligonucleotide primers were then combined by PCR using the cDNA template to generate a cyanomoloous monkey Neutrokine-alpha nucleic acid molecule. 5' primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3' primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36) . In the degenerate primer sequences (SEQ ID NO: 35 and 36), "I" stands for deoxyinosine or dideoxyinosine.

In another embodiment, the polynucleotide of the present invention comprises or consists of the sequence shown in SEQ ID NO: 29, and/or the sequence encoding the amino acid sequence shown in SEQ ID NO: 30, fragments, variants and derivatives thereof . These polynucleotides are also encompassed within the scope of the present invention. For example, some embodiments of the present invention relate to polynucleotides comprising or having at least 80%, 85%, 90%, 92%, 95%, 96%, 97 %, 98% or 99% identity to the sequence composition of the polypeptide sequences. The amino acid sequence derived from the translation of SEQ ID NO:29 is shown in SEQ ID NO:30. Fragments, variants and derivatives comprising or consisting of the amino acid sequence shown in SEQ ID NO: 30, and the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30 are also encompassed by the present invention. For example, some embodiments of the present invention relate to polypeptides comprising or consisting of at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% of amino acids 68-219 of SEQ ID NO: 30 % or 99% identical polypeptide sequences. The nucleic acid molecule having the sequence shown in SEQ ID NO: 29 was obtained from rhesus monkey PBMCs by RT-PCR using two degenerate primers. Briefly, total RNA was prepared from rhesus monkey PBMCs by using Trizol (purchased from Life Technologies, Inc, Rockville, MD) according to the manufacturer's instructions. Single-stranded cDNA was then synthesized by standard methods from the rhesus monkey PBMC preparation using the oligo dT primer. Neutrokine-α-specific primers are designed based on the conserved region between mouse and human Neutrokine-α molecules (SEQ ID NO: 22 and 1, respectively). Then, the cynomolgus Neutrokine-α nucleic acid molecule was generated by PCR using the cDNA template in combination with the following two degenerate oligonucleotide primers. 5' primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3' primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequences of the degenerate primers (SEQ ID NO: 35 and 36), "I" stands for deoxyinosine or dideoxyinosine.

The present invention also provides a nucleic acid molecule having a nucleotide sequence related to an extension of SEQ ID NO: 1 and SEQ ID NO: 18, which nucleotide sequence has been determined from the following related cDNA clone: HSOAD 55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8), and HLTBM08 (SEQ ID NO: 9).

The invention further relates to nucleic acid molecules encoding a portion of the nucleotide sequences described herein, as well as fragments of such isolated nucleic acid molecules. In one embodiment, the present invention provides a polynucleotide having a nucleotide sequence representing a portion of SEQ ID NO: 1, which consists of nucleotides 1-1001 of SEQ ID NO: 1. In another embodiment, the present invention provides a polynucleotide having a nucleotide sequence representing a portion of SEQ ID NO: 18 consisting of nucleotides 1-798 of SEQ ID NO: 18.

The invention further relates to fragments of the nucleic acid molecules (ie, polynucleotides) described herein. A nucleic acid molecule fragment having, for example, the following nucleotide sequence refers to a length of at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40, 50, 100, 150, 200, 250, 300, 325 , 350, 375, 400, 450, or 500 nucleotides, the nucleotide sequence is for example: the nucleotide sequence of the cDNA contained in the plasmid of ATCC deposit number 97768, encoded by the plasmid of ATCC deposit number 97768 The nucleotide sequence of the polypeptide sequence encoded by the cDNA contained, the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the polypeptide sequence encoding SEQ ID NO: 2, the cDNA contained in the plasmid of ATCC deposit number 203518 Nucleotide sequence, the nucleotide sequence of SEQ ID NO: 18, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 20, or their complementary strands. These fragments have many uses including, but not limited to, diagnostic probes and primers as described herein. Of course, larger fragments such as fragments of 501-1500 nucleotides in length are also useful according to the invention, which fragments correspond to most, if not all, of the following nucleotide sequences: contained in the plasmid of ATCC deposit number 97768 The nucleotide sequence of the cDNA, the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the cDNA contained in the plasmid of ATCC Accession No. 203518, and the nucleotide sequence of SEQ ID NO: 18. Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a polypeptide comprising or represented by Neutrokine-α shown in Figures 1A and 1B (SEQ ID NO: 2) and Figures 5A and 5B (SEQ ID NO: 19), respectively and/or the epitope-carrying portion of the Neutrokine-αSV polypeptide, and see details below. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention.

A nucleic acid molecule fragment having, for example, the following nucleotide sequence refers to a length of at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40, 50, 100, 150, 200, 250, 300, 325 , 350, 375, 400, 450 or 500 nucleotides, said nucleotide sequence is for example: the nucleotide sequence of SEQ ID NO: 21, the nucleotide sequence of SEQ ID NO: 22, SEQ ID NO: The nucleotide sequence of 27, the nucleotide sequence of SEQ ID NO: 29, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 23, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 28, encoding SEQ ID NO: 28 The nucleotide sequence of the polypeptide sequence of ID NO: 30 or their complementary strands. These fragments have many uses including, but not limited to, use as diagnostic probes and primers as described herein. Of course, larger fragments such as fragments of 501-1500 nucleotides in length are also useful according to the invention, such fragments corresponding to most, if not all, of the following nucleotide sequences: nucleotides of SEQ ID NO: 21 Acid sequence, the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence of SEQ ID NO:27, the nucleotide sequence of SEQ ID NO:29, the nucleotide sequence of the polypeptide sequence encoding SEQ ID NO:23 , the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 28, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 30, or their complementary strands. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention.

Representative fragments of Neutrokine-alpha polynucleotide fragments of the present invention include, for example, comprising or consisting of about 1-50, 51-100, 101-146 of the cDNA of SEQ ID NO: 1 or its complementary strand or ATCC deposit number 97768 , 147-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 600-650, 651-700, 701-750, 751 -A fragment consisting of 800, 800-850, 851-900, 901-950, 951-1000, 1001-1050 and/or 1051-1082 nucleotide sequences. As used herein, "about" includes the specified range, as well as ranges that are a few (5, 4, 3, 2, or 1) nucleotides more or less than the range at either or both ends.

Representative fragments of Neutrokine-αSV polynucleotide fragments of the present invention include, for example, comprising or consisting of SEQ ID NO: 18 or its complementary strand, or about 1-50, 51-100, 101- 146, 147-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, and/or 851-900 nucleotide sequences. "About" herein includes the specified range, as well as ranges that are a few (5, 4, 3, 2, or 1) nucleotides more or less than the range at either or both ends.

In some preferred embodiments, the polynucleotide of the present invention comprises or consists of 571-627,580-627,590-627,600-627,610-627,571-620,580-620 of SEQ ID NO:1 , 590-620, 600-620, 571-610, 580-610, 590-610, 571-600, 580-600 and/or 571-590 nucleotide residues.

In certain other preferred embodiments, the polynucleotide of the present invention comprises or consists of the following nucleotide residues of SEQ ID NO: 18: 1-879, 25-879, 50-879, 75-879, 100- 879, 125-879, 150-879, 175-879, 200-879, 225-879, 250-879, 275-879, 300-879, 325-879, 350-879, 375-879, 400-879, 425-879, 450-879, 475-879, 500-879, 525-879, 550-879, 575-879, 600-879, 625-879, 650-879, 675-879, 700-879, 725- 879, 750-879, 775-879, 800-879, 825-879, 850-879, 1-850, 25-850, 50-850, 75-850, 100-850, 125-850, 150-850, 175-850, 200-850, 225-850, 250-850, 275-850, 300-850, 325-850, 350-850, 375-850, 400-850, 425-850, 450-850, 475- 850, 500-850, 525-850, 550-850, 575-850, 600-850, 625-850, 650-850, 675-850, 700-850, 725-850, 750-850, 775-850, 800-850, 825-850, 1-825, 25-825, 50-825, 75-825, 100-825, 125-825, 150-825, 175-825, 200-825, 225-825, 250- 825, 275-825, 300-825, 325-825, 350-825, 375-825, 400-825, 425-825, 450-825, 475-825, 500-825, 525-825, 550-825, 575-825, 600-825, 625-825, 650-825, 675-825, 700-825, 725-825, 750-825, 775-825, 800-825, 1-800, 25-800, 50- 800, 75-800, 100-800, 125-800, 150-800, 175-800, 200-800, 225-800, 250-800, 275-800, 300-800, 325-800, 350-800, 375-800, 400-800, 425-800, 450-800, 475-800, 500-800, 525-800, 550-800, 575-800, 600-800, 625-800, 650-800, 675- 800, 700-800, 725-800, 750-800, 775-800, 1-775, 25-775, 50-775, 75-775, 100-775, 125-775, 150-775, 175-775, 200-775, 225-775, 250-775, 275-775, 300-775, 325-775, 350-775, 375-775, 400-775, 425-775, 450-775, 475-775, 500- 775, 525-775, 550-775, 575-775, 600-775, 625-775, 650-775, 675-775, 700-775, 725-775, 750-775, 1-750, 25-750, 50-750, 75-750, 100-750, 125-750, 150-750, 175-750, 200-750, 225-750, 250-750, 275-750, 300-750, 325-750, 350- 750, 375-750, 400-750, 425-750, 450-750, 475-750, 500-750, 525-750, 550-750, 575-750, 600-750, 625-750, 650-750, 675-750, 700-750, 725-750, 1-725, 25-725, 50-725, 75-725, 100-725, 125-725, 150-725, 175-725, 200-725, 225- 725, 250-725, 275-725, 300-725, 325-725, 350-725, 375-725, 400-725, 425-725, 450-725, 475-725, 500-725, 525-725, 550-725, 575-725, 600-725, 625-725, 650-725, 675-725, 700-725, 1-700, 25-700, 50-700, 75-700, 100-700, 125- 700, 150-700, 175-700, 200-700, 225-700, 250-700, 275-700, 300-700, 325-700, 350-700, 375-700, 400-700, 425-700, 450-700, 475-700, 500-700, 525-700, 550-700, 575-700, 600-700, 625-700, 650-700, 675-700, 1-675, 25-675, 50- 675, 75-675, 100-675, 125-675, 150-675, 175-675, 200-675, 225-675, 250-675, 275-675, 300-675, 325-675, 350-675, 375-675, 400-675, 425-675, 450-675, 475-675, 500-675, 525-675, 550-675, 575-675, 600-675, 625-675, 650-675,1- 650, 25-650, 50-650, 75-650, 100-650, 125-650, 150-650, 175-650, 200-650, 225-650, 250-650, 275-650, 300-650, 325-650, 350-650, 375-650, 400-650, 425-650, 450-650, 475-650, 500-650, 525-650, 550-650, 575-650, 600-650, 625- 650, 1-625, 25-625, 50-625, 75-625, 100-625, 125-625, 150-625, 175-625, 200-625, 225-625, 250-625, 275-625, 300-625, 325-625, 350-625, 375-625, 400-625, 425-625, 450-625, 475-625, 500-625, 525-625, 550-625, 575-625, 600- 625, 1-600, 25-600, 50-600, 75-600, 100-600, 125-600, 150-600, 175-600, 200-600, 225-600, 250-600, 275-600, 300-600,325-600,350-600,375-600,400-600,425-600,450-600,475-600,500-600,525-600,550-600,575-600,1- 575, 25-575, 50-575, 75-575, 100-575, 125-575, 150-575, 175-575, 200-575, 225-575, 250-575, 275-575, 300-575, 325-575, 350-575, 375-575, 400-575, 425-575, 450-575, 475-575, 500-575, 525-575, 550-575, 1-550, 25-550, 50- 550, 75-550, 100-550, 125-550, 150-550, 175-550, 200-550, 225-550, 250-550, 275-550, 300-550, 325-550, 350-550, 375-550, 400-550, 425-550, 450-550, 475-550, 500-550, 525-550, 1-525, 25-525, 50-525, 75-525, 100-525, 125- 525, 150-525, 175-525, 200-525, 225-525, 250-525, 275-525, 300-525, 325-525, 350-525, 375-525, 400-525, 425-525, 450-525, 475-525, 500-525, 1-500, 25-500, 50-500, 75-500, 100-500, 125-500, 150-500, 175-500, 200-500, 225- 500, 250-500, 275-500, 300-500, 325-500, 350-500, 375-500, 400-500, 425-500, 450-500, 475-500, 1-475, 25-475, 50-475, 75-475, 100-475, 125-475, 150-475, 175-475, 200-475, 225-475, 250-475, 275-475, 300-475, 325-475, 350- 475, 375-475, 400-475, 425-475, 450-475, 1-450, 25-450, 50-450, 75-450, 100-450, 125-450, 150-450, 175-450, 200-450, 225-450, 250-450, 275-450, 300-450, 325-450, 350-450, 375-450, 400-450, 425-450, 1-425, 25-425, 50- 425, 75-425, 100-425, 125-425, 150-425, 175-425, 200-425, 225-425, 250-425, 275-425, 300-425, 325-425, 350-425, 375-425, 400-425, 1-400, 25-400, 50-400, 75-400, 100-400, 125-400, 150-400, 175-400, 200-400, 225-400, 250- 400, 275-400, 300-400, 325-400, 350-400, 375-400, 1-375, 25-375, 50-375, 75-375, 100-375, 125-375, 150-375, 175-375, 200-375, 225-375, 250-375, 275-375, 300-375, 325-375, 350-375, 1-350, 25-350, 50-350, 75-350, 100- 350, 125-350, 150-350, 175-350, 200-350, 225-350, 250-350, 275-350, 300-350, 325-350, 1-325, 25-325, 50-325, 75-325, 100-325, 125-325, 150-325, 175-325, 200-325, 225-325, 250-325, 275-325, 300-325, 1-300, 25-300, 50- 300, 75-300, 100-300, 125-300, 150-300, 175-300, 200-300, 225-300, 250-300, 275-300, 1-275, 25-275, 50-275, 75-275, 100-275, 125-275, 150-275, 175-275, 200-275, 225-275, 250-275, 1-250, 25-250, 50-250, 75-250, 100- 250, 125-250, 150-250, 175-250, 200-250, 225-250, 1-225, 25-225, 50-225, 75-225, 100-225, 125-225, 150-225, 175-225, 200-225, 1-200, 25-200, 50-200, 75-200, 100-200, 125-200, 150-200, 175-200, 1-175, 25-175, 50- 175, 75-175, 100-175, 125-175, 150-175, 1-150, 25-150, 50-150, 75-150, 100-150, 125-150, 1-125, 25-125, 50-125, 75-125, 100-125, 1-100, 25-100, 50-100, 75-100, 1-75, 25-75, 50-75, 1-50, 25-50, and/or or 1-25

In other preferred embodiments, the polynucleotide of the present invention comprises or consists of the following nucleotide residues of SEQID NO: 1: 400-627, 425-627, 450-627, 475-627, 500-627 , 525-627, 550-627, 575-627, 600-627, 400-600, 425-600, 450-600, 475-600, 500-600, 525-600, 550-600, 575-600, 400 -575, 425-575, 450-575, 475-575, 500-575, 525-575, 550-575, 400-550, 425-550, 450-550, 475-550, 500-550, 525-550 , 400-500, 425-500, 450-500, 475-500, 400-475, 425-475, 450-475, 400-450, 425-450, 571-800, 600-800, 625-800, 650 -800, 675-800, 700-800, 725-800, 750-800, 775-800, 571-775, 600-775, 625-775, 650-775, 675-775, 700-775, 725-775 , 750-775, 571-750, 600-750, 625-750, 650-750, 675-750, 700-750, 725-750, 571-725, 600-725, 625-725, 650-725, 675 -725, 700-725, 571-700, 600-700, 625-700, 650-700, 675-700, 571-675, 600-675, 625-675, 650-675, 571-650, 600-650 , 625-650, 571-625, 600-625, and/or 571-600

In other preferred embodiments, the polynucleotide of the present invention comprises or consists of the following nucleotide residues of SEQID NO: 1: 147-500, 147-450, 147-400, 147-350, 200-500 , 200-450, 200-400, 200-350, 250-500, 250-450, 250-400, 250-350, 300-500, 300-450, 300-400, 300-350, 350-750, 350 -700, 350-650, 350-600, 350-550, 400-750, 400-700, 400-650, 400-600, 400-550, 425-750, 425-700, 425-650, 425-600 , 425-550, 450-1020, 450-1001, 450-950, 450-900, 450-850, 450-800, 450-775, 500-1001, 500-950, 500-900, 500-850, 500 -800, 500-775, 550-1001, 550-950, 550-900, 550-850, 550-800, 550-775, 600-1001, 600-950, 600-900, 600-850, 600-800 , 600-775, 650-1001, 650-950, 650-900, 650-850, 650-800, 650-775, 700-1001, 700-950, 700-900, 700-850, 700-800, 700 -775, 825-1082, 850-1082, 875-1082, 900-1082, 925-1082, 950-1082, 975-1082, 1000-1082, 1025-1082, and/or 1050-1082

Preferably, the polynucleotide fragment of the present invention encodes a polypeptide exhibiting Neutrokine-α and/or Neutrokine-αSV functional activity. A polypeptide having "functional activity" refers to a polypeptide that exhibits one or more known functional activities associated with full-length and/or secreted Neutrokine-alpha polypeptides and/or Neutrokine-alphaSV polypeptides. Such functional activity includes, but is not limited to, biological activity (such as the ability to stimulate B cell proliferation, survival, differentiation, and/or activation), antigenicity (binding (or binding to Neutrokine-α and/or Neutrokine-αSV polypeptide competitively) anti-Neutrokine - the ability of alpha and/or anti-Neutrokine-αSV antibodies], immunogenicity (the ability to produce antibodies binding to Neutrokine-α and/or Neutrokine-αSV polypeptides), formation with Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention Ability to multimerize, and bind to Neutrokine-α and/or Neutrokine-αSV polypeptide receptors or ligands.

In other specific embodiments, the polypeptide encoded by the polynucleotide fragment of the present invention comprises or is deduced from the intracellular domain of Neutrokine-α (1-46 amino acids of SEQ ID NO: 2), the transmembrane domain (SEQ ID NO: 2) deduced ID NO: 47-72 amino acids of 2), putative ectodomain (73-284 amino acids of SEQ ID NO: 2), putative TNF conserved domain (191-284 amino acids of SEQ ID NO: 2). In another embodiment, a polynucleotide fragment of the invention comprises or consists of any combination of 1, 2, 3 or all 4 of the above domains. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another specific embodiment, the polypeptide encoded by the polynucleotide fragment of the present invention comprises or consists of the putative intracellular domain of Neutrokine-αSV (1-46 amino acids of SEQ ID NO: 19), the putative transmembrane domain (SEQ ID NO: 47-72 amino acids of 19), putative ectodomain (73-266 amino acids of SEQ ID NO: 19), or putative TNF conserved domain (SEQ ID NO: 172-265 amino acids of 19 )composition. In other embodiments, the polypeptide encoded by the polynucleotide fragment of the present invention comprises or consists of any combination of 1, 2, 3 or all 4 of the above domains. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises or consists of a polynucleotide encoding Met1-Lys113, Leu114-Thr141, Ile142-Lys160, Amino acid sequences of Gly161-Gln198, Val199-Ala248 and Gly250-Leu285. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises or consists of a polynucleotide encoding Met1-Lys113, Leu114-Thr141, Gly142-Gln179, Amino acid sequence of Val180-Ala229, Gly230-Leu266 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises or consists of a polynucleotide encoding Met 1-Lys106, Leu107-Thr134, Glu135-Asn165, Amino acid sequences of Ile167-Lys184, Gly185-Gln224, Val225-Ala272 and Gly273-Leu309 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises or consists of a polynucleotide encoding Tyr 1-Lys47, Leu48-Thr75, Ile76-Lys94 selected from SEQ ID NO: 28, Amino acid sequences of Gly95-Gln132, Val133-Ala182 and Gly183-Ala219 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises or consists of a polynucleotide encoding Tyr 1-Lys47, Leu48-Thr75, Ile76-Lys94 selected from SEQ ID NO: 30, Amino acid sequences of Gly95-Gln132, Val 133-Ala182 and Gly183-Ala219 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide of the present invention comprises or consists of the sequence shown in SEQ ID NO:21. The sequence shown in SEQ ID NO: 21 encodes a polypeptide, which consists of the initial methionine residue and the Ala134-Leu285 residues of the connected Neutrokine-α polypeptide sequence shown in SEQ ID NO: 2. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In certain other preferred embodiments, the polynucleotide of the present invention comprises or consists of the following nucleotide residues of SEQ ID NO: 21: 1-459, 15-459, 30-459, 45-459, 60- 459, 75-459, 90-459, 105-459, 120-459, 135-459, 150-459, 165-459, 180-459, 195-459, 210-459, 225-459, 240-459, 255-459, 270-459, 285-459, 300-459, 315-459, 330-459, 345-459, 360-459, 375-459, 390-459, 405-459, 420-459, 435- 459, 450-459, 1-450, 15-450, 30-450, 45-450, 60-450, 75-450, 90-450, 105-450, 120-450, 135-450, 150-450, 165-450, 180-450, 195-450, 210-450, 225-450, 240-450, 255-450, 270-450, 285-450, 300-450, 315-450, 330-450, 345- 450, 360-450, 375-450, 390-450, 405-450, 420-450, 435-450, 1-435, 15-435, 30-435, 45-435, 60-435, 75-435, 90-435, 105-435, 120-435, 135-435, 150-435, 165-435, 180-435, 195-435, 210-435, 225-435, 240-435, 255-435, 270- 435, 285-435, 300-435, 315-435, 330-435, 345-435, 360-435, 375-435, 390-435, 405-435, 420-435, 1-420, 15-420, 30-420, 45-420, 60-420, 75-420, 90-420, 105-420, 120-420, 135-420, 150-420, 165-420, 180-420, 195-420, 210- 420, 225-420, 240-420, 255-420, 270-420, 285-420, 300-420, 315-420, 330-420, 345-420, 360-420, 375-420, 390-420, 405-420, 1-405, 15-405, 30-405, 45-405, 60-405, 75-405, 90-405, 105-405, 120-405, 135-405, 150-405, 165- 405, 180-405, 195-405, 210-405, 225-405, 240-405, 255-405, 270-405, 285-405, 300-405, 315-405, 330-405, 345-405, 360-405, 375-405, 390-405, 1-390, 15-390, 30-390, 45-390, 60-390, 75-390, 90-390, 105-390, 120-390, 135- 390, 150-390, 165-390, 180-390, 195-390, 210-390, 225-390, 240-390, 255-390, 270-390, 285-390, 300-390, 315-390, 330-390, 345-390, 360-390, 375-390, 1-375, 15-375, 30-375, 45-375, 60-375, 75-375, 90-375, 105-375, 120- 375, 135-375, 150-375, 165-375, 180-375, 195-375, 210-375, 225-375, 240-375, 255-375, 270-375, 285-375, 300-375, 315-375, 330-375, 345-375, 360-375, 1-360, 15-360, 30-360, 45-360, 60-360, 75-360, 90-360, 105-360, 120- 360, 135-360, 150-360, 165-360, 180-360, 195-360, 210-360, 225-360, 240-360, 255-360, 270-360, 285-360, 300-360, 315-360, 330-360, 345-360, 1-345, 15-345, 30-345, 45-345, 60-345, 75-345, 90-345, 105-345, 120-345, 135- 345, 150-345, 165-345, 180-345, 195-345, 210-345, 225-345, 240-345, 255-345, 270-345, 285-345, 300-345, 315-345, 330-345, 1-330, 15-330, 30-330, 45-330, 60-330, 75-330, 90-330, 105-330, 120-330, 135-330, 150-330, 165- 330, 180-330, 195-330, 210-330, 225-330, 240-330, 255-330, 270-330, 285-330, 300-330, 315-330, 1-315, 15-315, 30-315, 45-315, 60-315, 75-315, 90-315, 105-315, 120-315, 135-315, 150-315, 165-315, 180-315, 195-315, 210- 315, 225-315, 240-315, 255-315, 270-315, 285-315, 300-315, 1-300, 15-300, 30-300, 45-300, 60-300, 75-300, 90-300, 105-300, 120-300, 135-300, 150-300, 165-300, 180-300, 195-300, 210-300, 225-300, 240-300, 255-300, 270- 300, 285-300, 1-285, 15-285, 30-285, 45-285, 60-285, 75-285, 90-285, 105-285, 120-285, 135-285, 150-285, 165-285, 180-285, 195-285, 210-285, 225-285, 240-285, 255-285, 270-285, 1-270, 15-270, 30-270, 45-270, 60- 270, 75-270, 90-270, 105-270, 120-270, 135-270, 150-270, 165-270, 180-270, 195-270, 210-270, 225-270, 240-270, 255-270, 1-255, 15-255, 30-255, 45-255, 60-255, 75-255, 90-255, 105-255, 120-255, 135-255, 150-255, 165- 255, 180-255, 195-255, 210-255, 225-255, 240-255, 1-240, 15-240, 30-240, 45-240, 60-240, 75-240, 90-240, 105-240, 120-240, 135-240, 150-240, 165-240, 180-240, 195-240, 210-240, 225-240, 1-225, 15-225, 30-225, 45- 225, 60-225, 75-225, 90-225, 105-225, 120-225, 135-225, 150-225, 165-225, 180-225, 195-225, 210-225, 1-210, 15-210, 30-210, 45-210, 60-210, 75-210, 90-210, 105-210, 120-210, 135-210, 150-210, 165-210, 180-210, 195- 210, 1-195, 15-195, 30-195, 45-195, 60-195, 75-195, 90-195, 105-195, 120-195, 135-195, 150-195, 165-195, 180-195, 1-180, 15-180, 30-180, 45-180, 60-180, 75-180, 90-180, 105-180, 120-180, 135-180, 150-180, 165- 180, 1-165, 15-165, 30-165, 45-165, 60-165, 75-165, 90-165, 105-165, 120-165, 135-165, 150-165, 1-150, 15-150, 30-150, 45-150, 60-150, 75-150, 90-150, 105-150, 120-150, 135-150, 1-135, 15-135, 30-135, 45- 135, 60-135, 75-135, 90-135, 105-135, 120-135, 1-120, 15-120, 30-120, 45-120, 60-120, 75-120, 90-120, 105-120, 1-105, 15-105, 30-105, 45-105, 60-105, 75-105, 90-105, 1-90, 15-90, 30-90, 45-90, 60- 90, 75-90, 1-75, 15-75, 30-75, 45-75, 60-75, 1-60, 15-60, 30-60, 45-60, 1-45, 15-45, 30-45, 1-30, and 1/or 15-30. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

Accordingly, specific embodiments of the present invention relate to polynucleotides encoding a polypeptide comprising or consisting of the beta sheet regions A, A', B, B', C, D, E shown in Figure 7A and Example 6 , F, G or H amino acid sequence composition. Other embodiments of the present invention relate to polynucleotides encoding Neutrokine-alpha polypeptides comprising or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of Figure 7A and Example 6 Any combination of β-sheet regions A-H is shown. Another preferred embodiment of the present invention relates to a polypeptide comprising or consisting of the β sheet region A, A', B, B', C, D, E, F, G or H shown in Figure 7A and Example 6 Neutrokine-α amino acid sequence composition. Additional embodiments of the present invention relate to Neutrokine-alpha polypeptides, which comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the ten beta sheet regions A-H shown in Figure 7A and Example 6. any combination.

In some other preferred embodiments, the polynucleotide of the present invention comprises or consists of 34-57, 118-123, 133-141, 151-159, 175-216, 232-255, 280- of SEQ ID NO: 21 315, 328-357, 370-393 and/or 430-456 nucleotide residues. These polynucleotides and polypeptides correspond to the putative beta-sheet regions shown in Figure 7A. In some embodiments, the polynucleotides of the present invention comprise or consist of at least 90 % 95%, 96%, 97%, 98% or 99% identical polynucleotide composition. The invention also encompasses the aforementioned polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotide sequences are also encompassed by the present invention. In another embodiment, the present invention provides an isolated nucleic acid molecule comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the above-mentioned beta sheets of the present invention under stringent hybridization conditions. A polynucleotide to which a polynucleotide hybridizes. The "stringent conditions" used herein are as described above.

In another preferred embodiment, the polynucleotide of the present invention comprises or consists of 576-599,660-665,675-683,693-701,717-758,744-803,822-857, 870-899, 912-935, and/or 972-998 nucleotide residues. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet regions shown in Figure 7A.

In another preferred embodiment, the polynucleotide of the present invention comprises or consists of 457-462, 472-480, 490-498, 514-555, 571-600, 619-654, 667-696 of SEQ ID NO: 18, 699-732 and/or 769-795 nucleotide residues. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet regions shown in Figure 7A.

In another preferred embodiment, the polynucleotide of the present invention comprises or consists of 124-129, 139-147, 157-165, 181-222, 238-267, 286-321, 334-363 of SEQ ID NO: 22, 376-399 and/or 436-462 nucleotide residues. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet regions shown in Figure 7A. Polypeptides comprising or consisting of the amino acid sequence of any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of these regions are also encompassed by the invention.

The relative positions of several intron/exon boundaries of murine Neutrokine-α (SEQ ID NO: 22 and SEQ ID NO: 23) were determined based on sequence analysis of murine genomic DNA. The apparent second exon at the 5' end of the mouse Neutrokine-α genomic clone (previously referred to as "exon 2") consists of the sequence Tyr187-Gln222 shown in SEQ ID NO:23. The apparent third exon at the 5' end of the murine Neutrokine-α genomic clone (previously referred to as "exon 3") contained Val 223-Gly273 of the sequence shown in SEQ ID NO:23.

Accordingly, in one embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of Tyr187-Gln222 of SEQ ID NO:23. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the polynucleotide sequence encoding the above-mentioned mouse Neutrokine-α polypeptide % identity polynucleotide sequence composition. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of Val223-Gly273 residues of SEQ ID NO:23. The present invention also relates to a polynucleotide comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polynucleotide sequence encoding the aforementioned murine Neutrokine-alpha polypeptide Nucleic acid molecules composed of acid sequences. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In addition, the relative positions of the corresponding intron/exon boundaries in human Neutrokine-α (SEQ ID NO: 1 and SEQ ID NO: 2) were determined based on the sequence alignment of mouse and human Neutrokine-α polypeptides. The apparent second exon at the 5' end of human Neutrokine-α (also previously referred to as "exon 2") consists of Tyr163-Gln198 of the sequence shown in SEQ ID NO:2. The apparent third exon at the 5' end of human Neutrokine-α (also previously referred to as "exon 3") consists of Val 199-G1y249 of the sequence shown in SEQ ID NO:2.

Therefore, in one embodiment, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of the Tyr163-Gln198 amino acid sequence shown in SEQ ID NO:2. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the polynucleotide sequence encoding the above-mentioned Neutrokine-α polypeptide identical polynucleotide sequences. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of the Val199-Gly249 amino acid sequence of SEQ ID NO:2. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the polynucleotide sequence encoding the above-mentioned Neutrokine-α polypeptide identical polynucleotide sequences. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

The functional activity of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides, and fragments, variant derivatives, and analogs thereof, can be assayed by various methods described herein and well known in the art.

For example, in one embodiment, assays for binding to or competing with full-length Neutrokine-α and/or Neutrokine-αSV polypeptides for binding to anti-Neutrokine-α and/or anti-Neutrokine-αSV antibodies, or binding to Neutrokine-α receptors on B cells Various immunoassays known in the art, including but not limited to competitive and non-competitive assays using the following methods: radioimmunoassay, ELISA (enzyme-linked immunosorbent assay) Adsorption assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (labeled with e.g. colloidal gold, enzymes or radioactive isotopes), Western blots, precipitation reactions, agglutination assays ( Such as gel agglutination analysis, hemagglutination analysis), complement fixation analysis, immunofluorescence analysis, protein A analysis, and immunoelectrophoresis analysis, etc. In one embodiment, antibody binding is detected by detecting a label on the original antibody. In another embodiment, the primary antibody is detected by detecting the binding of the secondary antibody and reagent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. Many methods for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In another embodiment, when identifying Neutrokine-α and/or Neutrokine-αSV ligands, or evaluating the ability of polypeptide fragments, variants or derivatives of the present invention to multimerize, binding can be assayed, for example by the present invention Methods are well known in the art such as reducing or non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See Phizicky, E. et al., 1995, Microbiology Research 59:94-123. In another embodiment, the physiological relevance of Neutrokine-α and/or Neutrokine-αSV binding to its substrates is analyzed.

In addition, the assays described herein (eg, see Examples 6 and 7) and other assays known in the art can be routinely used to assay Neutrokine-α and/or Neutrokine-αSV polypeptides and fragments, variant derivatives and analogs thereof The ability to stimulate Neutrokine-α and/or Neutrokine-αSV-related biological activities (such as stimulating or inhibiting (in the case of Neutrokine-α and/or Neutrokine-αSV antagonists) B cell proliferation, differentiation and/or activation and/or Prolong B cell survival in vitro or in vivo).

Other methods are well known to those skilled in the art and are also encompassed by the present invention.

In additional embodiments, the polynucleotides of the invention encode polypeptides having the functional characteristics of Neutrokine-α and Neutrokine-αSV. Preferred embodiments of the invention for this purpose include fragments comprising or consisting of the following regions of Neutrokine-alpha and Neutrokine-alpha SV polypeptides: alpha helix and alpha helix forming region ("alpha region"), beta sheet and beta sheet forming region ("beta region"), corner and turn forming region ("turn region"), coil and curl forming region ("crimp region"), hydrophilic region, hydrophobic region, alpha amphipathic region, beta amphiphilic region, flexible region , the surface formation area and the high antigenic index area.

It is believed that one or more beta sheet regions of Neutrokine-alpha shown in Figure 7A are important for dimerization and the interaction between Neutrokine-alpha and its ligand.

Some preferred regions for this purpose are shown in Figure 3 (Table 1). The data shown in Figure 3 and Table 1 represent only different versions of the same results obtained when analyzing the amino acid sequence of SEQ ID NO: 2 with the default parameters of the DNA*STAR computer program.

The aforementioned preferred regions shown in Figure 3 and Table 1 include, but are not limited to, the aforementioned types of regions identified by analyzing the amino acid sequences shown in Figures 1A and 1B. As shown in Figure 3 and Table 1, such preferred regions include Garnier-Robson α region, β region, corner region, and coiled region, Chou-Fasman α region, β region and coiled region, Kyte-Doolittle hydrophilic region and Hydrophobic region, Eisenberg α and β amphipathic regions, Karplus-Schulz flexible region, Emini surface forming region, and Jameson-Wolf high antigenic index region. Highly preferred polynucleotides are those encoding polypeptides consisting of regions of Neutrokine-α and/or Neutrokine-αSV that combine some (eg 1, 2, 3, or 4) of the above features.

Additionally, the data in columns VIII, IX, XIII and XIV of Table 1 can be routinely used to determine regions of Neutrokine-α that are highly antigenically potentially antigenic (column VIII of Table 1 represents hydrophilicity according to Kyte-DooLittle; Table 1 Column IX of Table 1 represents hydrophobicity according to Hopp-Woods; Column XIII of Table 1 represents antigenic index according to Jameson-Wolf; Column XIV of Table 1 represents surface probability according to Emini). Regions of high antigenicity are determined from the data set forth in VIII, IX, XIII, and/or XIV by selecting values representing regions of the polypeptide that are susceptible to surface exposure of the polypeptide in an environment that occurs at the initiation of an immune response. The environment in which antigen recognition can occur. The data shown in Figure 6 can also be conventionally represented in a similar tabular form by simply detecting the amino acid sequence shown in Figure 6 (SEQ ID NO: 19) using the DNA*STAR model and program with default parameters set. As mentioned above, the amino acid sequence shown in Figure 6 can also be used to determine the region of Neutrokine-α with high potential antigenicity, which can be represented in the form of a diagram (such as Figure 6) or a table (such as Table 1).

Table 1

Residue position I II III IV V VI VI VII VIII IX X XI XII XIII XIV

Met 1 A . . . . . . . 0.73 -0.71 . . . 0.95 1.39

Asp 2 A . . . . . T . 1.12 -0.66 * . . 1.15 1.56

Asp 3 A . . . . . T . 1.62 -1.09 * . . 1.15 2.12

Ser 4 A . . . . . T . 2.01 -1.51 . . . 1.15 4.19

Thr 5 A . . . . . T . 2.40 -2.13 . . F 1.30 4.35

Glu 6 A A . . . . . . -2.70 -1.73 * * F 0.90 4.51

Arg 7 A A . . . . . . 2.81 -1.34 * * F 0.90 4.51

Glu 8 A A . . . . . . . 2.00 -1.73 * * F 0.90 6.12

Gln 9 A A . . . . . . . 1.99 -1.53 * * F 0.90 2.91

Ser 10 A . . B . . . 2.00 -1.04 * * F 0.90 2.15

Arg 11 A . . B . . . 1.33 -0.66 * * F 0.90 1.66

Leu 12 A . . B . . . 0.41 -0.09 * * F 0.45 0.51

Thr 13 A . . B . . . 0.46 0.20 * * F -0.15 0.32

Ser 14 A A . . . . . . 0.50 -0.19 * * . 0.30 0.32

Cys 15 A A A . . . . . . 0.91 -0.19 * * . 0.30 0.78

Leu 16 A A . . . . . . 0.80 -0.87 * * F 0.90 1.06

Lys 17 A A . . . . . . 1.61 -1.36 . * F 0.90 1.37

Lys 18 A A . . . . . . 1.32 -1.74 . * F 0.90 4.44

Arg 19 A A . . . . . . 1.67 -1.70 . * F 0.90 5.33

Glu 20 A A . . . . . . 1.52 -2.39 . * F 0.90 5.33

Glu 21 A A . . . . . . 2.38 -1.70 . * F 0.90 2.20

Mel 22 A A . . . . . . 2.33 -1.70 . * F 0.90 2.24

Lys 23 A A . . . . . . 1.62 -1.70 * * F 0.90 2.24

Leu 24 A A . . . . . . 0.66 -1.13 * * F 0.75 0.69

Lys 25 A A . . . . . . 0.36 -0.49 . * F 0.45 0.52

Glu 26 A A . B . . . -0.53 -0.71 * * . 0.60 0.35

Cys 27 A A . B . . . . -0.74 -0.03 * * . 0.30 0.30

Val 28 A A . B . . . . -1.00 -0.03 * * . 0.30 0.12

Ser 29 A A . B . . . -0.08 0.40 * * . -0.30 0.11

Ile 30 A . . B . . . -0.08 0.40 * * . -0.30 0.40

Leu 31 A . . B . . . -0.08 -0.17 * . . 0.45 1.08

Pro 32 . . . . B . . C 0.29 -0.81 * . F 1.10 1.39

Arg 33 . . . . . T . . 0.93 -0.81 . * F 1.50 2.66

Lys 34 . . . . . T . . 0.93 -1.07 . . F 1.84 4.98

Glu 35 . . . . . . . C 0.97 -1.37 * * F 1.98 4.32

Ser 36 . . . . . . T C 1.89 -1.16 * * F 2.52 1.64

Pro 37 . . . . . . T C 1.80 -1.16 * * F 2.86 1.60

Ser 38 . . . . . T T . 1.39 -0.77 * . F 3.40 1.24

Val 39 A . . . . . T . 1.39 -0.39 . * F 2.36 1.24

Arg 40 A . . . . . . . 1.39 -0.77 * * F 2.46 1.60

Ser 41 A . . . . . . . . 1.34 -1.20 * * F 2.46 2.00

Ser 42 . . . . . T T . 1.60 -1.16 . * F 3.06 2.67

Lys 43 . . . . . T T . 1.09 -1.80 . * F 3.06 2.72

Asp 44 . . . . . T T T . 1.13 -1.11 * * F 3.40 1.67

Gly 45 A . . . . . T . 0.43 -0.81 * * F 2.66 1.03

Lys 46 A A . . . . . . 0.14 -0.70 . . F 1.77 0.52

Leu 47 A A . . . . . . 0.13 -0.20 * . . 0.98 0.31

Leu 48 A A . . . . . . . -0.72 0.29 * . . 0.04 0.46

Ala 49 A A . . . . . . -1.53 0.54 . * . -0.60 0.19

Ala 50 A A A . . . . . . -2.00 1.23 . . . -0.60 0.19

Thr 51 A A . . . . . . -2.63 1.23 . . . -0.60 0.19

Leu 52 A A . . . . . . -2.63 1.04 . . . -0.60 0.19

Leu 53 A A . . . . . . -2.63 1.23 . . . -0.60 0.15

Leu 54 A A . . . . . . -2.34 1.41 . . . -0.60 0.09

Ala 55 A A A . . . . . . -2.42 1.31 . . . -0.60 0.14

Leu 56 A A . . . . . . -2.78 1.20 . . . -0.60 0.09

Leu 57 A . . . . . T . -2.78 1.09 . . . -0.20 0.06

Table 1 (continued)

Residue position I I II III IV V V VI VII VIII IX X XI XII XIII XIV

Set 58 A . . . . . T . -2.28 1.09 . . . -0.20 0.05

Cys 59 A . . . . . T . -2.32 1.07 . . . -0.20 0.09

Cys 60 A . . . . . T . -2.59 1.03 . . . -0.20 0.08

Leu 61 . . B B B . . . -2.08 0.99 . . . -0.60 0.04

Thr 62 . . B B B . . . -1.97 0.99 . . . -0.60 0.11

Val 63 . . B B B . . . -1.91 1.20 . . . -0.60 0.17

Val 64 . . B B B . . . -1.24 1.39 . . . -0.60 0.33

Ser 65 . . B B B . . . -1.43 1.10 . . . -0.60 0.40

Phe 66 A . . . B . . . . -1.21 1.26 . . . -0.60 0.40

Tyr 67 A . . . B . . . . -1.49 1.11 . . . -0.60 0.54

Gln 68 A . . . B . . . -1.44 0.97 . . . -0.60 0.41

Val 69 A . . . B . . . . -0.59 1.27 . . . -0.60 0.39

Ala 70 A . . . B . . . . -0.63 0.89 . . . -0.60 0.43

Ala 71 A . . B . . . 0.07 0.56 . * . -0.60 0.25

Leu 72 A . . . . . T . -0.50 0.16 . * . 0.10 0.55

Gln 73 A . . . . . T . -1.09 0.20 . . F 0.25 0.45

Gly 74 A . . . . . T . -0.53 0.20 . . F 0.25 0.45

Asp 75 A . . . . . T . -0.76 0.09 . * F 0.25 0.73

Leu 76 A A . . . . . . . -0.06 0.09 . * F -0.15 0.35

Ala 77 A A A . . . . . . 0.17 -0.31 . * . 0.30 0.69

Ser 78 A A . . . . . . 0.17 -0.24 . * . 0.30 0.42

Leu 79 A A . . . . . . . -0.30 -0.24 . * . 0.30 0.88

Arg 80 A A . . . . . . . -0.30 -0.24 . * . 0.30 0.72

Ala 81 A A . . . . . . 0.17 -0.34 . * . 0.30 0.93

Glu 82 A A . . . . . . 0.72 -0.30 . * . 0.45 1.11

Leu 83 A A . . . . . . . 0.99 -0.49 . * . 0.30 0.77

Gln 84 A A . . . . . . . 1.21 0.01 . * * . -0.15 1.04

Gly 85 A A . . . . . . . 1.10 0.01 * * * . -0.30 0.61

His 86 A A . . . . . . . 1.73 0.01 * * * . -0.15 1.27

His 87 A A . . . . . . 0.92 -0.67 . * . 0.75 1.47

Ala 88 A A . . . . . . . 1.52 -0.39 . * . 0.45 1.22

Glu 89 A A . . . . . . 0.93 -0.39 . . . 0.45 1.39

Lys 90 A A . . . . . . 0.93 -0.39 * . F 0.60 1.03

Leu 91 A . . . . . . T . 0.38 -0.46 * . . 0.85 1.01

Pro 92 A . . . . . T . 0.07 -0.46 . . . 0.70 0.59

Ala 93 A . . . . . T . 0.07 -0.03 . . . 0.70 0.29

Gly 94 A . . . . . T . -0.14 0.47 . . . -0.20 0.36

Ala 95 A . . . . . . . . -0.14 0.21 . * . -0.10 0.36

Gly 96 A . . . . . . . 0.08 -0.21 . . F 0.65 0.71

Ala 97 A . . . . . . . . -0.06 -0.21 . . F 0.65 0.72

Pro 98 A . . . . . . . . -0.28 -0.21 . * F 0.65 0.71

Lys 99 A A . . . . . . 0.07 -0.03 . . F 0.45 0.59

Ala 100 A A A . . . . . . 0.66 -0.46 . . F 0.60 1.01

Gly 101 A A . . . . . . 0.41 -0.96 . . F 0.90 1.13

Leu 102 A A . . . . . . 0.79 -0.89 . . F 0.75 0.57

Glu 103 A A . . . . . . 0.41 -0.46 * . F 0.45 0.88

Glu 104 A A . . . . . . . -0.49 -0.46 * . F 0.45 0.89

Ala 105 A A . . . . . . . -0.21 -0.24 . . . 0.30 0.81

Pro 106 A A . . . . . . . -0.46 -0.44 . . . 0.30 0.67

Ala 107 A A A . . . . . . 0.01 0.06 . . . -0.30 0.39

Val 108 A A . . . . . . . -0.80 0.49 . * . -0.60 0.38

Thr 109 A A . . . . . . . -0.76 0.67 . * . -0.60 0.20

Ala 110 A A . . . . . . . -1.06 0.24 * * * . -0.30 0.40

Gly 111 A A . . . . . . . -1.54 0.43 * * * . -0.60 0.38

Leu 112 A A . . . . . . . -0.96 0.57 * * * . -0.60 0.23

Lys 113 . A B . . . . . -0.31 0.09 * * * . -0.30 0.39

Ile 114 . A B . . . . . -0.21 0.01 * . . -0.30 0.61

Table 1 (continued)

Residue position I I II III IV V VI VII VIII IX X XI XII XIII XIV

Phe 115 . A B . . . . . -0.21 0.01 * . . 0.15 1.15

Glu 116 . A . . . . . C -0.08 -0.17 * . F 1.25 0.58

Pro 117 . A . . . . . C 0.39 0.26 * * * F 1.10 1.28

Pro 118 . . . . . . . C 0.34 -0.00 . . F 2.20 1.47

Ala 119 . . . . . . T C 0.89 -0.79 . * F 3.00 1.47

Pro 120 . . . . . . T C 1.59 -0.36 . * F 2.25 0.94

Gly 121 . . . . . T T T . 1.29 -0.39 . * F 2.15 0.98

Glu 122 . . . . . T T T . 1.20 -0.43 . . F 2.00 1.30

Gly 123 . . . . . . . . C 1.41 -0.54 . . F 1.60 1.12

Asn 124 . . . . . . T C 2.00 -0.57 . . F 1.50 1.97

Ser 125 . . . . . . T C 1.91 -0.60 . * F 1.50 1.82

Ser 126 . . . . . . T C 2.37 -0.21 . * F 1.54 2.47

Gln 127 . . . . . . T C 2.37 -0.64 . * F 2.18 3.01

Asn 128 . . . . . . . . C 2.76 -0.64 . . F 2.32 3.61

Ser 129 . . . . . . T C 2.87 -1.03 . . F 2.86 5.39

Arg 130 . . . . . T T T . 2.58 -1.41 * . F 3.40 6.09

Asn 131 . . . . . T T T . 2.02 -1.31 * . F 3.06 3.83

Lys 132 . . . . . T T T . 2.02 -1.07 * . F 2.72 2.12

Arg 133 . . . . . T . . . 1.68 -1.06 * . F 2.18 1.88

Ala 134 . . . . . . . . C 1.77 -0.63 * . F 1.64 1.15

Val 135 . . . . . . . C 1.66 -0.60 * . F 1.49 0.89

Gln 136 . . . . . . . . C 1.66 -0.60 * . F 1.83 0.79

Gly 137 . . . . . . T C 1.30 -0.60 * . F 2.52 1.35

Pro 138 . . . . . . T C 0.33 -0.61 * . F 2.86 2.63

Glu 139 . . . . . T T T . 0.61 -0.61 * . F 3.40 1.13

Glu 140 A . . . . . T . 1.47 -0.53 * . F 2.66 1.64

Thr 141 A . . . . . . . 1.47 -0.56 . . F 2.12 1.84

Val 142 A . . . . . . . . 1.14 -0.99 . . F 1.78 1.77

Thr 143 A . . . . . T . 0.54 -0.41 . . F 1.19 0.55

Gln 144 A . . . . . T . 0.54 0.27 * . F 0.25 0.31

Asp 145 A . . . . . T . -0.27 0.19 * . F 0.25 0.73

Cys 146 A . . . . . T . -0.84 0.23 * . . 0.10 0.42

Leu 147 A A A . . . . . . -0.58 0.43 * . . -0.60 0.17

Gln 148 A A . . . . . . . -0.27 0.53 * . . -0.60 0.10

Leu 149 A A . . . . . . . -0.57 0.53 * * * . -0.30 0.32

Ile 150 A A A . . . . . . . -0.57 0.34 * . . 0.30 0.52

Ala 151 . A . . . . . C -0.21 -0.34 . * . 1.40 0.52

Asp 152 . . . . . T T T . 0.39 -0.26 . * F 2.45 0.91

Ser 153 . . . . . . T C 0.08 -0.51 . . F 3.00 2.00

Glu 154 . . . . . . T C -0.00 -0.71 . . F 2.70 2.86

Thr 155 . . . . . . T C 0.89 -0.53 * . F 2.40 1.20

Pro 156 . . . . B . . . C 1.52 -0.13 * . F 1.56 1.55

Thr 157 . . . . B T . . 1.18 -0.51 * . F 1.92 1.79

Ile 158 A . . . B . . . 1.18 -0.09 . . F 1.08 1.23

Gln 159 . . . . . T T T . 0.93 -0.19 . . F 2.04 1.07

Lys 160 . . . . . T T T . 0.93 0.14 * . F 1.60 1.16

Gly 161 . . . . . T T T . 0.44 0.14 * . F 1.44 2.38

Ser 162 . . . . . T T T . -0.10 0.24 * . F 1.28 1.19

Tyr 163 . . . . B T . . 0.58 0.49 * . . 0.12 0.44

Thr 164 . . . B B . . . 0.29 0.91 * . . -0.44 0.69

Phe 165 . . B B B . . . -0.57 1.40 * . . -0.60 0.54

Val 166 . . . B B . . . -1.03 1.70 . . . -0.60 0.29

Pro 167 . . . B B . . . -1.03 1.63 . . . -0.60 0.16

Trp 168 A . . . B . . . -1.49 1.53 . * * . -0.60 0.25

Leu 169 A . . . B . . . . -1.13 1.53 * . . -0.60 0.29

Leu 170 A . . . B . . . . -0.32 0.89 * . . -0.30 0.38

Table 1 (continued)

Residue position I I II III IV V VI VI VII VIII IX X XI XII XIII XIV

Ter 171 A . . . . . . . 0.19 0.46 * . . 0.20 0.71

Phe 172 . . . . . T . . . 0.10 -0.03 * . . 1.80 0.85

Lys 173 . . . . . T T T . -0.20 -0.33 * . F 2.60 1.38

Arg 174 . . . . . . T C -0.20 -0.51 . . F 3.00 1.04

Gly 175 . . . . . . . T C 0.61 -0.21 . . F 2.25 0.99

Ser 176 A . . . . . T . 0.91 -1.00 * . F 2.05 0.86

Ala 177 A A A . . . . . . . 1.66 -1.00 * . F 1.35 0.76

Leu 178 A A . . . . . . . 1.61 -1.00 . . F 1.20 1.54

Glu 179 A A . . . . . . . 1.50 -1.43 . . F 0.90 1.98

Glu 180 A A . . . . . . . 1.89 -1.41 * . F 0.90 3.16

Lys 181 A A . . . . . . . 1.30 -1.91 * . F 0.90 7.66

Glu 182 A A . . . . . . . 1.08 -1.91 . . F 0.90 3.10

Asn 183 A A . . . . . . . 1.03 -1.23 * * F 0.90 1.48

Lys 184 A A . . . . . . . 1.08 -0.59 * . F 0.75 0.55

Ile 185 A A . . . . . . . 1.08 -0.59 * * * . 0.60 0.63

Leu 186 A A . . . . . . 0.72 -0.59 * * * . 0.60 0.68

Val 187 A A . . . . . . 0.38 -0.50 . * . 0.30 0.49

Lys 188 A A . . . . . . . 0.13 -0.07 * * F 0.45 0.69

Glu 189 A . . . . . T . -0.61 0.00 * * * F 0.40 1.32

Thr 190 . . . . . T T T . -0.42 0.10 . * F 0.80 1.54

Gly 191 . . . . . T T T . -0.50 0.24 * . F 0.65 0.67

Tyr 192 . . . . . T T T . 0.11 0.93 * * * . 0.20 0.27

Phe 193 . . . B B . . . -0.28 1.69 . . . -0.60 0.29

Phe 194 . . B B B . . . -0.28 1.63 . * . -0.60 0.29

Ile 195 . . . B B . . . -0.82 1.60 . . . -0.60 0.32

Tyr 196 . . . B B B . . . -1.29 1.49 . . . -0.60 0.28

Gly 197 . . . . B T . . . -1.29 1.39 . . . -0.20 0.26

Gln 198 . . . . B T . . -0.90 1.36 . . . -0.20 0.59

Val 199 . . . . B . . C -0.20 1.16 . . . -0.40 0.54

Leu 200 . . . . B . . C 0.73 0.40 . . . -0.10 0.92

Tyr 201 . . . . . . T T T . 0.67 -0.03 . . . 1.25 1.06

Thr 202 . . . . . T T T . 0.77 0.06 . . F 0.80 2.06

Asp 203 . . . . . T T T . 0.18 0.17 . . F 0.80 3.91

Lys 204 A . . . . . T . 0.43 -0.01 . . F 1.00 2.52

Thr 205 A A . . . . . . 0.90 -0.16 . . F 0.60 1.73

Tyr 206 A A . . . . . . . 1.11 -0.21 . . . 0.45 1.03

Ala 207 A A A . . . . . . . 0.61 0.29 . . . -0.30 0.70

Met 208 A A A . . . . . . . -0.28 0.97 . . . -0.60 0.40

Gly 209 A A A . B . . . . -0.32 1.17 * . . -0.60 0.18

His 210 A A A . B . . . 0.10 0.81 * . . -0.60 0.31

Leu 211 A A A . B . . . 0.39 0.31 . . . -0.30 0.61

Ile 212 A A A . B . . . 1.02 -0.30 . . . 0.45 1.22

Gln 213 A A . B . . . 0.77 -0.73 . * . 0.75 1.80

Arg 214 A A . B . . . . 1.08 -0.59 . * F 0.90 1.62

Lys 215 A A A . B B . . . 0.26 -0.77 * * F 0.90 3.14

Lys 216 A A . B . . . 0.37 -0.81 . * F 0.90 1.35

Val 217 . A B B . . . 0.91 -0.43 * * * . 0.30 0.60

His 218 . A B B . . . 0.91 -0.00 . * . 0.30 0.29

Val 219 . A B B B . . . 0.80 -0.00 * * * . 0.30 0.25

Phe 220 . . . B B . . . -0.06 -0.00 * . . 0.30 0.57

Gly 221 A . . . B . . . . -0.40 0.04 . * . -0.30 0.35

Asp 222 A . . . . . . . . -0.36 -0.07 * . . 0.50 0.63

Glu 223 A . . . . . . . -1.18 -0.03 * . . 0.50 0.60

Leu 224 A . . . B . . . . -0.63 -0.17 . . . 0.30 0.45

Ser 225 A . . . B . . . . -0.74 -0.11 . . . 0.30 0.39

Leu 226 A . . . B . . . . -1.10 0.57 . * . -0.60 0.18

Val 227 A . . . B . . . . -0.99 1.36 . * . -0.60 0.19

Table 1 (continued)

Residue position I I II III IV V V VI VII VIII IX X XI XII XIII XIV

Thr 228 A . . . B . . . . -1.66 0.67 * * * . -0.60 0.28

Leu 229 A . . . B . . . . -1.73 0.86 * . . -0.60 0.18

Phe 230 A . . . B . . . -1.43 0.86 * . . -0.60 0.17

Arg 231 A . . . B . . . -0.62 0.61 * . . -0.60 0.21

Cys 232 . . . . B T . . -0.37 0.53 * . . -0.20 0.41

Ile 233 . . . . B T . . . -0.27 0.46 * . . . -0.20 0.46

Gln 234 . . . . B T . . 0.54 0.10 * . . 0.10 0.37

Asn 235 . . . . B . . C 0.93 0.10 * . . 0.05 1.19

Met 236 . . . . B . . C 0.01 0.01 * . F 0.20 2.44

Pro 237 . . . . B . . C 0.47 0.01 * . F 0.44 1.16

Glu 238 . . . . . T . . . 1.36 0.04 * . F 1.08 1.12

Thr 239 . . . . . . . C 1.36 0.04 * . F 1.12 1.82

Leu 240 . . . . . . . . C 1.06 -0.17 * . F 1.96 1.89

Pro 241 . . . . . T . . 0.99 -0.21 . . F 2.40 1.46

Asn 242 . . . . . T . . 0.96 0.36 . . F 1.41 0.54

Asn 243 . . . . . . T T T . 0.66 0.63 . . F 1.22 1.03

Ser 244 . . . . . T T T . 0.38 0.33 . . F 1.13 0.89

Cys 245 . . . . . T T T . 0.84 0.40 . . . 0.74 0.56

Tyr 246 . . . . . T T T . 0.17 0.43 . . . 0.20 0.35

Ser 247 A . . . . . . . -0.42 0.71 . . . -0.40 0.18

Ala 248 A A . . . . . . . -0.38 0.83 . . . -0.60 0.34

Gly 249 A A A . . . . . . . -0.89 0.26 . . . -0.30 0.43

Ile 250 A A A . . . . . . . -0.22 0.19 * . . -0.30 0.27

Ala 251 A A A . . . . . . . 0.02 -0.20 * . . 0.30 0.46

Lys 252 A A . . . . . . . -0.02 -0.70 . . . 0.60 0.80

Leu 253 A A . . . . . . 0.57 -0.70 . . F 0.90 1.13

Glu 254 A A . . . . . . 0.91 -1.39 . . F 0.90 1.87

Glu 255 A A . . . . . . 0.99 -1.89 . . F 0.90 1.62

Gly 256 A A . . . . . . . 1.58 -1.20 . * F 0.90 1.62

Asp 257 A A . . . . . . 0.72 -1.49 . * F 0.90 1.62

Glu 258 A A . . . . . . . 0.94 -0.80 * * F 0.75 0.77

Leu 259 A A . . . . . . 0.06 -0.30 * * . 0.30 0.79

Gln 260 A A . . . . . . . -0.16 -0.04 * . . 0.30 0.33

Leu 261 A A . . . . . . . 0.30 0.39 * . . -0.30 0.30

Ala 262 A A A . . . . . . . 0.30 0.39 * . . -0.30 0.70

Ile 263 A A . . . . . . 0.30 -0.30 . * . 0.30 0.70

Pro 264 A . . . . . T . 0.52 -0.30 . * F 1.00 1.37

Arg 265 A . . . . . T . 0.52 -0.49 . * F 1.00 1.37

Glu 266 A . . . . . T . 0.44 -0.59 * * F 1.30 3.38

Asn 267 A . . . . . T . 0.73 -0.59 * * * F 1.30 1.53

Ala 268 A . . . . . . . 0.81 -0.63 * * . 0.95 1.05

Gln 269 A . . . . . . . . 1.02 0.06 * * * . -0.10 0.50

Ile 270 A . . . . . . . 0.57 0.06 . * . 0.15 0.52

Ser 271 . . . . . . . C 0.57 0.09 . * . 0.60 0.51

Leu 272 . . . . . . . C -0.29 -0.41 . * F 1.60 0.49

Asp 273 . . . . . T T T . -0.01 -0.17 . * F 2.25 0.52

Gly 274 . . . . . . T T T . -0.71 -0.37 . * F 2.50 0.56

Asp 275 . . . . . T T T . -0.52 0.03 . * F 1.65 0.59

Val 276 A . . . . . T . -0.57 0.13 . * F 1.00 0.30

Thr 277 A . . . B . . . -0.34 0.56 . * . -0.10 0.30

Phe 278 A . . . B . . . -1.16 0.63 . * . -0.35 0.18

Phe 279 A . . . B . . . . -0.77 1.31 . * . -0.60 0.20

Gly 280 A A A . . . . . . . -1.58 0.67 . * . -0.60 0.28

Ala 281 A A A . . . . . . . -1.53 0.87 . * . -0.60 0.27

Leu 282 A A . . . . . . . -1.61 0.77 * . . -0.60 0.26

Lys 283 A A . . . . . . . -1.30 0.41 * . . -0.60 0.33

Leu 284 A A . . . . . . -0.99 0.41 . . . -0.60 0.42

Leu 285 A A A . . . . . . . -1.03 0.34 * . . -0.30 0.65

Other preferred nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of sequences encoding one or more epitope-bearing portions of Neutrokine-alpha. In particular, such nucleic acid fragments of the present invention include or are composed of nucleic acid molecules encoding sequences selected from the following polypeptides: about Phe115-Leu147, Ile150-Tyr163, Ser170-Phe194, Glu223-Tyr246, Ser271- Phe278. As used herein, "about" refers to the specified range and this range is more or less 5, 4, 3, 2, or 1 amino acid residues at one or both of the amino and carboxy termini. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention. The polypeptide fragment carrying the antigenic epitope of Neutrokine-α can be easily determined by those skilled in the art by the above-mentioned Jameson-Wolf antigenic index analysis, as shown in FIG. 3 . Methods for determining other such epitope-carrying portions of Neutrokine-α are described in detail below.

Other preferred nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of sequences encoding one or more epitope-carrying portions of Neutrokine-αSV. Especially this nucleic acid fragment of the present invention comprises or is made up of the nucleic acid molecule of the sequence that is selected from following polypeptide coding: about Pro32-Leu47 of SEQ ID NO:19 aminoacid sequence, Glu116-Ser143, Phe153-Tyr173, Pro218-Tyr227, Ser252 -Thr258, Ala232-Gln241, Ile244-Ala249, Ser252-Val257. As used herein, "about" refers to the specified range, and this range is more or less 5, 4, 3, 2, or 1 amino acid residues at one or both of the amino-terminal and carboxyl-terminal ends. Polypeptide fragments carrying Neutrokine-α antigenic epitopes can be easily determined by those skilled in the art by the above-mentioned Jameson-Wolf antigenic index analysis. Methods for determining other such epitope-bearing portions of Neutrokine-αSV are described in detail below.

In specific embodiments, the polynucleotides of the invention are less than 100,000kb, 50,000kb, 10,000kb, 1,000kb, 500kb, 400kb, 350kb, 300kb, 250kb, 200kb, 175kb, 150kb, 125kb, 100kb, 75kb, 50kb in length , 40kb, 30kb, 25kb, 20kb, 15kb, 10kb, 7.5kb, or 5kb.

In another embodiment, the polynucleotide of the present invention comprises at least 15, at least 30, at least 50, at least 100 or at least 250, at least 500, at least 1000 contiguous nucleotides of the Neutrokine-alpha coding sequence, but defined by Less than or equal to 1000kb, 500kb, 250kb, 200kb, 150kb flanking the 5' or 3' coding nucleotides shown in 1A and 1B (SEQ ID NO: 1) or Figures 5A and 5B (SEQ ID NO: 18), Genomic DNA composition of 100kb, 75kb, 50kb, 30kb, 25kb, 20kb, 15kb, 10kb or 5kb. In another embodiment, a polynucleotide of the invention comprises at least 15, at least 30, at least 50, at least 100 or at least 250, at least 500, or at least 1000 contiguous nucleotides of the Neutrokine-α coding sequence, but does not comprise All or part of any Neutrokine-alpha intron. In another embodiment, the nucleic acid comprising the Neutrokine-alpha coding sequence does not contain the coding sequences of the genomic flanking genes (ie, the 5' or 3' sequences of the Neutrokine-alpha gene in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequences of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2 or 1 genomic flanking gene.

In another embodiment, the present invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent conditions to a portion of the polynucleotides in the above-mentioned nucleic acid molecule of the present invention, for example: complementary to FIGS. 1A and 1B ( The coding and/or non-coding sequence shown in SEQ ID NO: 1), the sequence of the cDNA clone of ATCC deposit number 97768, complementary to the coding and/or non-coding sequence shown in Figure 5A and 5B (SEQ ID NO: 18) , the sequence of the cDNA clone deposited at ATCC Accession No. 203518, complementary to the coding and/or non-coding sequence shown in SEQ ID NO: 21, (i.e. transcribed, untranslated) sequence, complementary to SEQ ID NO: 22 The sequence of the coding and/or non-coding sequence shown is complementary to the coding and/or non-coding sequence shown in SEQ ID NO:27 The sequence is complementary to the coding and/or non-coding sequence shown in SEQ ID NO:29 sequences, or fragments of these sequences (eg open reading frames or fragments thereof). "Stringent hybridization conditions" means incubation overnight at 42°C followed by rinsing the filter at approximately 65°C with 0.1xSSC in a solution containing: 50% formamide, 5xSSC (750mM NaCl, 75mM lemon trisodium phosphate), 50mM sodium phosphate (pH7.6), 5X Denhard's solution, 10% dextran sulfate, and 20μg/ml denatured sheared salmon sperm DNA

A polynucleotide that hybridizes to a "part" of a polynucleotide refers to a polynucleotide with a length of at least 15, preferably at least 20, more preferably at least 30, most preferably at least 30 to 70 (such as 40, 50, or 60), especially Any integer number of nucleotides in the range of 30 to 70 or 80 to 150 is preferred, and a polynucleotide (DNA or RNA) hybridized to a full-length reference polynucleotide is even preferred. Uses of these polynucleotides include, but are not limited to, use as diagnostic probes and primers, and are described in detail below. A portion of a polynucleotide of "at least about 20 nucleotides" in length, for example, is meant to include the specified range. Greater or less than 5, 4, 3, 2, 1 or 0 amino acids at one or both ends of the nucleotide sequence of the reference polynucleotide (such as either or both sequences of the two deposited cDNAs, Figures 1A and 1B ( The complementary strand of the nucleotide sequence shown in SEQ ID NO: 1), the complementary strand of the nucleotide sequence shown in Figure 5A and 5B (SEQ ID NO: 18), the complementary strand of the nucleotide sequence shown in SEQ ID NO: 21 strand, the complementary strand of the nucleotide sequence shown in SEQ ID NO:22, the complementary strand of the nucleotide sequence shown in SEQ ID NO:27, and/or the complementary strand of the nucleotide sequence shown in SEQ ID NO:29) . Of course, polynucleotides that only hybridize to the following sequences are not included in the polynucleotides used in the present invention to hybridize to a portion of the nucleic acids of the present invention, because such polynucleotides will hybridize to any poly(A) sequence or its complementary sequence. Nucleic acid molecules (such as, virtually any double-stranded cDNA clone produced with oligo dT as a primer), said sequence is a poly(A) sequence (3 of Neutrokine-α cDNA as shown in Figures 1A and 1B (SEQ ID NO: 1) ' terminal poly(A) sequence segment, the 3' terminal poly(A) sequence segment of Neutrokine-αSV cDNA shown in Figure 5A and 5B (SEQ ID NO:18), or the 3 of Neutrokine-αSV cDNA shown in SEQ IDNO:22 'terminal poly(A) sequence segment), or a segment of T (or U) residue complementary sequence.

As indicated above, nucleic acid molecules of the present invention encoding Neutrokine-α or Neutrokine-αSV polypeptides may include, but are not limited to: polynucleotides that themselves encode the amino acid sequence of the ectodomain of the corresponding polypeptide; the coding sequence of the ectodomain of the corresponding polypeptide and additional sequences , such as the sequence encoding the intracellular domain and the transmembrane domain sequence, or a preprotein, proprotein or preproprotein sequence; the coding sequence of the corresponding extracellular domain of the polypeptide with or without the aforementioned additional coding sequence.

The nucleic acids of the invention also encode the above protein sequences with additional non-coding sequences, such as but not limited to introns and non-coding 5' and 3' sequences, such as in transcription, mRNA processing including splicing and polyadenylation signals Examples include transcribed, untranslated sequences that play a role in chromosomal binding and mRNA stability; additional coding sequences that encode additional amino acids, such as those that provide additional functions.

Thus, a sequence encoding a polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In some preferred embodiments of the invention, the marker amino acid sequence is a 6-histidine peptide, such as the tags provided in pQE vectors (QIAGEN, Inc, 9259 Eton, Averue, Chatsworth, CA, 91311), many of which are commercially available. purchase. The 6-histidine allows for routine purification of fusion proteins as described by Gentz et al., Proc. The "HA" tag is another peptide used for purification that corresponds to an epitope derived from the influenza hemagglutinin protein as described by Wilson et al., Cell 37:767 (1984). Other such fusion proteins include Neutrokine-[alpha] or Neutrokine-[alpha]SV polypeptide fused to Fc at the N- or C-terminus, as described below.

The present invention also relates to variants of the nucleic acid molecules of the present invention, which encode a part, analog or derivative of the Neutrokine-α or Neutrokine-αSV polypeptide of SEQ ID NO:2. A variant may be naturally occurring, such as a natural allelic variant. An "allelic" variant refers to one of several variations of a gene occupying a given locus on a chromosome of an organism. See Gene II, Lewin, B.ed, John Wiley & Sons, New York (1985). Non-naturally occurring variants can be generated by mutagenesis methods known in the art, including but not limited to oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, e.g., Carter et al., Nucleic Acids Res. 13:4331 (1986); and Zoller et al., Nucleic Acids Res. 10:6487 (1982), Cassette Mutagenesis (see, for example, Wells et al., Gene 34:315 (1985)), Restricted Selection Mutagenesis (see, for example, Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).

Such variants include those produced by nucleotide substitutions, deletions or additions. Such substitutions, deletions or additions may comprise one or more amino acids. A variant may be a change in a coding region, a non-coding region, or both. Changes within the coding region may result in conservative or non-conservative amino acid substitutions, deletions or additions. Particularly preferred among these are silent substitutions, additions and deletions which do not alter the properties and activities of Neutrokine-α and/or Neutrokine-αSV polypeptides or a part thereof. Also particularly preferred are conservative amino acid substitutions.

Other embodiments of the present invention relate to isolated nucleic acid molecules comprising a polynucleotide encoding the amino acid sequence of a Neutrokine-α and/or Neutrokine-αSV polypeptide comprising at least 1 but not more than 50, preferably No more than 40, more preferably no more than 30, especially preferably no more than 20, 10-20, 5-10, 1-5, 3-5, or 1-3 conservative amino acid substitutions. Of course, the preferred sequence is polynuclear amino acid sequences encoding Neutrokine-α and/or Neutrokine-αSV polypeptides containing no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitution. glycosides.

Other embodiments include isolated nucleic acid molecules comprising or consisting of at least 80%, 85% or 90% identity, preferably at least 95%, 96%, 97%, 98% identity to a polynucleotide selected from the group consisting of, Or the polynucleotide composition of the nucleotide sequence of 99% identity: (a) coding has the nucleus of the Neutrokine-alpha polypeptide of complete aminoacid sequence shown in Figure 1A and 1B (being 1-285 position of SEQ ID NO:2) Nucleotide sequence; (b) encoding has the nucleotide sequence of the Neutrokine-alpha polypeptide shown in Figure 1A and 1B except the complete amino acid sequence (i.e. the 2-285 amino acids of SEQ ID NO: 2) except the N-terminal methionine (c) a fragment of (b) polypeptide with Neutrokine-α functional activity (such as antigenicity or biological activity); (d) encoding has the 73-285 amino acid sequence in Fig. 1A and 1B (SEQ ID NO: 2) The nucleotide sequence of the putative extracellular domain of Neutrokine-alpha polypeptide; (e) the nucleotide sequence of the Neutrokine-alpha polypeptide coding has 134-285 aminoacid sequence among Fig. 1A and 1B (SEQ ID NO:2); ( f) a nucleotide sequence encoding a Neutrokine-α polypeptide, which has a complete amino acid sequence encoded by a cDNA clone deposited with ATCC Accession No. 97768; (g) a nucleotide sequence encoding the ectodomain of a Neutrokine-α polypeptide, the polypeptide has the amino acid sequence encoded by the cDNA deposited with ATCC Accession No. 97768; and (h) is complementary to (a), (b), (c), (d), (e), (f), (g) above or The nucleotide sequence of any nucleotide sequence of (h). The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also encompassed by the present invention.

Particularly preferred embodiments of the present invention relate to nucleic acid molecules comprising or having at least 80%, 85%, 90% identity, preferably at least 95%, 96%, 97% identity to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide , 98%, 99% or 100% identical nucleotide sequence polynucleotide composition, the Neutrokine-α polypeptide has the 134-285 amino acid sequence in Figures 1A and 1B (SEQ ID NO: 2). Preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 90% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having 134-285 amino acid sequence in Fig. 1A and 1B (SEQ ID NO:2). A more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide, said Neutrokine-alpha polypeptide It has the amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence at least 96% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide It has the amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2).

In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence at least 97% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide, said Neutrokine-alpha polypeptide The alpha polypeptide has the amino acid sequence 134-285 in Figures 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence at least 98% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide It has the amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2). More preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide It has the amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2).

Another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a polynucleotide, the amino acid sequence of which polynucleotide encodes a Neutrokine-αSV polypeptide (as described herein Neutrokine-αSV polypeptide fragment) contains at least 1 but no more than 50, Preferably no more than 40, more preferably no more than 30, most preferably no more than 20 conservative amino acid substitutions. Of course, a particularly preferred polynucleotide is one that encodes the amino acid sequence of a Neutrokine-α polypeptide, having a sequence containing no more than 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, Amino acid sequence with 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions.

Other embodiments include isolated nucleic acid molecules comprising or consisting of polynucleotides having at least 80%, 85%, 90% identity, preferably at least 95%, 96 %, 97%, 98%, or 99% identical nucleotide sequence: (a) encodes a Neutrokine-αSV polypeptide having the complete amino acid sequence shown in Figure 5A and 5B (ie, 1-266 of SEQ ID NO: 19) Nucleotide sequence; (b) coding has the nucleoside of the Neutrokine-αSV polypeptide shown in Figure 5A and 5B except the complete amino acid sequence (ie 2-266 of SEQ ID NO: 19) except the N-terminal methionine Acid sequence; (c) coding has the nucleotide sequence of the deduced extracellular domain of Neutrokine-αSV polypeptide having 73-285 amino acid sequence in 5A and 5B (SEQ ID NO:19); (d) coding Neutrokine-αSV polypeptide The nucleotide sequence of the Neutrokine-αSV polypeptide has the complete amino acid sequence encoded by the cDNA clone deposited at ATCC Accession No. 203518; (e) the nucleotide sequence encoding the extracellular domain of the Neutrokine-αSV polypeptide has the The amino acid sequence encoded by the cDNA clone at ATCC Deposit No. 203518; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e) above .

In addition, polynucleotides encompassed by the present invention comprise or are at least about 10, 20, 25 or 30 of the 1-1082 nucleotide sequences in Figures 1A and 1B (SEQ ID NO: 1) or are at least 90% or 95% identical. Contiguous nucleotides, preferably at least about 40 or 50 contiguous amino acids of any part of the sequence, preferably excluding the nucleotide sequences determined from the above four cDNA clones and 797-1082, 810-1082, and 346-542 bit nucleotide sequence. The present invention also includes a polynucleotide comprising or consisting of about 10, 20, 25 or 30 contiguous nucleotides that are at least 90% or 95% identical to the sequence shown in Figures 5A and 5B (SEQ ID NO: 18) , preferably at least about 40 or 50 nucleotides in any part of the sequence, preferably excluding the nucleotide sequences determined from the above four cDNA clones. The present invention also includes a polynucleotide comprising or consisting of at least 90% or 95% identical to about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40 or 50, of the sequence shown in SEQ ID NO: 21. The sequence composition of any part of consecutive nucleotides preferably does not include the nucleotide sequences determined from the above four cDNA clones. The present invention also includes a polynucleotide comprising or consisting of at least 90% or 95% identical to about 10, 20, 25 or 30 contiguous nucleotides of the sequence shown in SEQ ID NO: 22, preferably at least about 40 or The sequence composition of any part of 50 contiguous nucleotides preferably does not include the nucleotide sequences determined from the above 4 cDNA clones. The present invention also includes a polynucleotide comprising or consisting of at least about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40, of the sequence shown in SEQ ID NO: 27 by at least 90% or 95% Or the sequence composition of any part of 50 consecutive nucleotides, preferably excluding the nucleotide sequences determined from the above four cDNA clones. The present invention also includes a polynucleotide comprising or consisting of at least about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40, of the sequence shown in SEQ ID NO: 29 by at least 90% or 95% Or the sequence composition of any part of 50 consecutive nucleotides, preferably excluding the nucleotide sequences determined from the above four cDNA clones. As used herein, "about" includes the specified range, as well as more or less (ie, 5, 4, 3, 2 or 1) amino acids at one or both ends.

A polynucleotide having a nucleotide sequence having, e.g., at least 95% "identity" to a reference nucleotide sequence encoding Neutrokine-α and/or Neutrokine-αSV polypeptide refers to the nucleotide sequence of this polynucleotide and The reference sequence is identical except that the polynucleotide sequence may include more than 5 mismatches per 100 nucleotides of the reference sequence encoding Neutrokine-α and/or Neutrokine-αSV polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference sequence, approximately 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or account for 5% of the reference sequence. Nucleotides of 5% of the total number of nucleotides in the sequence can be inserted into the reference sequence. These mutations in the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere in between these terminal positions, scattered at individual nucleotides or one or more contiguous groups in the reference sequence between. The reference (reference) sequence can be the entire nucleotide sequence encoding Neutrokine-α and/or Neutrokine-αSV, as shown in Figures 1A and 1B (SEQ ID NO: 1) and Figures 5A and 5B (SEQ ID NO: 18) respectively Shown, or any Neutrokine-α such as SEQ ID NO:21,22,27,28 shown Neutrokine-α polynucleotide, or any Neutrokine-α or Neutrokine-αSV polynucleotide fragments described herein.

In fact, is any particular nucleic acid molecule at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide sequences shown in Figures 1A and 1B, or Figure 5A and 5B, or the nucleotide sequence of a deposited cDNA clone, or any Neutrokine-α polynucleotide such as the Neutrokine-α polynucleotide shown in SEQ ID NO: 21, 22, 27 or 28 or its Fragments can be determined routinely using known computer programs (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Seience Drive, Madison, WI 53711). Bestfit uses the local homologous sequence alignment program of Smith and Waterman to find the best segment of homology between two sequences (Advances in Applied Mathematics 2:482-489 (1981)). When using Bestfit or any other sequence comparison program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention, parameters are of course set such that the percent identity is calculated over the full-length reference nucleotide sequence, And the homologous gaps accounting for 5% of the total number of nucleotides in the reference sequence are allowed.

In a specific embodiment, the identity between the reference (reference) sequence (sequence of the invention) and the subject sequence, also called global sequence alignment, is determined using the FAS TDB computer based on the sequence alignment of Brutlag and colleagues. Determined by the procedure (Comp. App. Biosci 6:237-245 (1990)). In a sequence alignment, both the reference sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The results of the overall sequence alignment are expressed as percent identity. Preferred parameters for FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the target nucleotide sequence, whichever is shorter. According to this embodiment, if the target sequence is shorter than the reference sequence because of a 5' or 3' deletion but not because of an intrinsic deletion, the results are manually adjusted because the FASTDB program does not take the target sequence into account when calculating percent identity5 ' and 3' truncated. For the target sequence truncated at the 5' and 3' ends relative to the reference sequence, calculate the number of unmatched/aligned bases of the reference sequence at the 5' and 3' of the target sequence as a percentage of the total bases of the reference sequence Percentage of base to adjust percent identity. Determining whether a nucleotide is a match/alignment is determined by the FASTDB sequence alignment results. This percentage is then subtracted from the percent identity calculated by the FASTDB program described above using the specified parameters to obtain the final percent identity. The result of this adjustment is used for the purposes of this embodiment. Only the bases that are not matched/aligned with the reference sequence outside the 5' and 3' bases of the target sequence by the FASTDB sequence alignment need to be calculated to manually adjust the percent identity results. For example, a 90-base subject sequence is aligned to a 100-base reference sequence to determine percent identity. The deletion occurs at the 5' end of the target sequence, so the FASTDB sequence alignment does not show a match/alignment of the first 10 bases at the 5' end. These 10 unpaired bases represent 10% of the sequence (number of unmatched bases at the 5' and 3' ends/total number of bases in the reference sequence), so this 10% should be calculated from the percent identity calculated by the FASTDB program Subtract from the result. If the remaining 90 bases are sufficiently matched, the final percent identity is 90%. In another embodiment, a 90-base target sequence is compared to a 100-base reference sequence. This deletion is an internal deletion, so there are bases not matched/aligned with the reference sequence at the 5' and 3' ends of the target sequence. In this case, the percent identity calculated by FASTDB does not require manual adjustment. Again, manual adjustments are only required if the 5' and 3' bases of the target sequence are not matched/aligned to the reference sequence. This implementation requires no other manual adjustments.

The present invention relates to nucleic acid molecules that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences (i.e., polynucleotides) disclosed herein, regardless of Whether they encode a polypeptide having Neutrokine-α and/or Neutrokine-αSV functional activity (such as biological activity), the nucleic acid sequence disclosed herein is, for example, the sequence shown in Figure 1A and 1B (SEQ ID NO: 1) or the cDNA of the deposit Nucleotide sequence. In addition, the present invention also relates to at least 80%, 85%, 90%, 92%, 95%, 96%, 97 %, 98% or 99% identical nucleic acid molecules, regardless of whether they encode a polypeptide having Neutrokine-αSV activity. In addition, the present invention also relates to at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence shown in SEQ ID NO: 21, 22, 27 or 28 Identical nucleic acid molecules, whether or not they encode a polypeptide having Neutrokine-α activity. This is because even if a specific nucleic acid molecule does not encode a polypeptide having Neutrokine-α and/or Neutrokine-αSV activity, those skilled in the art know how to use the nucleic acid molecule, for example, as a hybridization probe or a polymerase chain reaction (PCR) primer. The purposes of the nucleic acid molecule of the present invention that does not encode a polypeptide having Neutrokine-α and/or Neutrokine-αSV activity include: (1) separating Neutrokine-α and/or Neutrokine-αSV gene or its allelic variants in a cDNA library (2) In situ hybridization (such as "FISH") with metaphase chromosome smears to provide the exact chromosomal location of Neutrokine-α and/or Neutrokine-αSV genes, such as Verma et al., Basic Methods Manual for Human Chromosomes, Pergamon Press, described in New York (1988); and for use in Northern blot analysis to detect expression of Neutrokine-α and/or Neutrokine-αSVmRNT in specific tissues.

However, it is preferably at least 80%, 85 %, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical nucleic acid molecule, and it actually encodes a polypeptide having Neutrokine-α and/or Neutrokine-αSV functional activity (such as biological activity ) of the polypeptide. It is also preferred to have at least 80%, 85%, 90%, 92%, 95%, 96%, 97% of the nucleotide sequence shown in Figure 5A and 5B (SEQ ID NO: 18), or the nucleotide sequence of the cDNA deposited. %, 98% or 99% identical nucleic acid molecule, and it actually encodes a polypeptide having Neutrokine-α and/or Neutrokine-αSV polypeptide functional activity (such as biological activity). Also preferably having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, or 98% identity with the nucleic acid sequence shown in SEQ ID NO: 21, 22, 27, or 28 The nucleic acid molecule of the sequence, and it actually encodes a polypeptide having Neutrokine-α and/or Neutrokine-αSV polypeptide functional activity (such as biological activity).

"Polypeptides with Neutrokine-α polypeptide functional activity (such as biological activity)" and "polypeptides with Neutrokine-αSV polypeptide functional activity (such as biological activity)" refer to specific functional analysis (such as immunological or biological analysis) The activity of the extracellular domain or the full-length polypeptide of the Neutrokine-α and/or Neutrokine-αSV polypeptide of the present invention is substantially similar, but not necessarily the same, as determined in the present invention. For example, the functional activity of Neutrokine-α and/or Neutrokine-αSV polypeptides can be achieved by the polypeptide sequence forming a multimer with the complete Neutrokine-α and/or Neutrokine-αSV or the extracellular domain of Neutrokine-α and/or Neutrokine-αSV (such as homodimer and homotrimer), and the ability to bind Neutrokine-α and/or Neutrokine-αSV polypeptide ligands are determined. The functional activity of Neutrokine-α and/or Neutrokine-αSV polypeptides can also be determined by determining the ability of the polypeptides of the present invention to induce proliferation, differentiation or activation of lymphocytes (such as B cells) and/or prolong the survival time of B cells. These functional assays can be routinely performed using the methods described herein (see, eg, Example 6) and others known in the art. In addition, Neutrokine-alpha or Neutrokine-alpha SV polypeptides of the invention modulate cell proliferation, cytotoxicity, cell survival and cell death. The effect of proteins on certain cells can be determined by performing in vitro cell proliferation, cytotoxicity, cell survival and cell death assays using reagents well known in the art and commonly available for detection of cell replication and/or death. For example, many such analyzes of the activity of TNF-related proteins are found in various references. Briefly, such an analysis involves, for example, collecting human or animal (e.g., murine) cells and combining them with (1) a supernatant of a transfected host cell containing the Neutrokine-alpha protein (or candidate polypeptide), or (2) Control non-transfected host cell supernatants were pooled and, after a certain period of incubation, the effect on cell number or viability was determined. Such cell proliferation and/or survival modulating activity measurable in such assays can be used in the treatment of tumors, tumor metastases, infections, autoimmune diseases, inflammation and other immune-related diseases.

Neutrokine-α modulates cell proliferation and differentiation in a dose-dependent manner in the assays described above. Thus, preferred "polypeptides having Neutrokine-alpha polypeptide functional activity (eg, biological activity)" include polypeptides that also exhibit any of the cell-modulating (especially immunomodulatory) activities in the above assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be equivalent to that of a Neutrokine-alpha polypeptide, it is preferred that a "polypeptide having the functional activity of a Neutrokine-alpha polypeptide" exhibit substantially similar dose dependence in a given activity compared to a Neutrokine-alpha polypeptide (that is, the candidate polypeptide exhibits higher activity than the reference Neutrokine-α polypeptide, or not less than 25 times, preferably not less than 10 times the activity).

In certain preferred embodiments, "polypeptides having Neutrokine-α polypeptide functional activity (such as biological activity)" and "polypeptides having Neutrokine-αSV polypeptide functional activity (such as biological activity)" include also presenting Fig. 8A, 8B , 9A, 9B, 11, 10, 12A and 12B and any of the same B cell (or other cell) regulatory (especially immunomodulatory) activity polypeptides described in Example 6.

Like other members of the TNF family, Neutrokine-α appears to act on blood cells including, for example, monocytes, lymphocytes (eg, B cells) and neutrophils. Therefore, Neutrokine-α is active in inducing proliferation, differentiation and migration of these cells. This activity is used for immune enhancement or suppression, bone marrow protection, stem cell migration, control of acute and chronic inflammation, and treatment of leukemia. Methods for assaying this activity are known in the art. See, eg, Peters et al., Current Immunology 17:273 (1996); Young et al., J. Experimental Methods 182:1111 (1995); Caux et al., Nature 390:258 (1992); and Santiago-Schwarz et al., Advances in Experimental Methods in Biology 378:7 (1995).

Of course, due to the degeneracy of the genetic code, those skilled in the art will immediately recognize that a large number of nucleic acid molecules will encode polypeptides "having Neutrokine-alpha polypeptide functional activity (such as biological activity)", said nucleic acid molecules having at least 80%, 85 %, 90%, 92%, 95%, 96%, 97%, 98% or 99% are equivalent to the nucleic acid sequence contained in the cDNA clone contained in the deposit of ATCC deposit number 97768, or Figure 1A and 1B (SEQ ID NO : 1) The nucleic acid sequences shown, or the sequences of their fragments. Those skilled in the art will also immediately realize that a large number of nucleic acid molecules encoding "polypeptides having Neutrokine-αSV polypeptide functional activity (such as biological activity)" have the same nucleic acid sequence contained in the cDNA clone of ATCC deposit number 203518, or Nucleic acid sequences shown in Figures 5A and 5B (SEQ ID NO: 18), sequences having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity. In fact, since the degenerate variants of these nucleotide sequences all encode the same polypeptide, those skilled in the art would be aware of this even without the above comparative analysis. It is further recognized in the art that some of such nucleic acid molecules that are not degenerate variants also encode polypeptides having Neutrokine-[alpha] and/or Neutrokine-[alpha]SV activity. This is because those skilled in the art are well aware that amino acid substitutions have little or no significant effect on protein function (such as replacement of one aliphatic amino acid with another aliphatic amino acid), as detailed below.

Similarly, polynucleotides encoding polypeptides comprising all or part of the V142-K160 region of SEQ ID NO: 2 appear to be of diagnostic and therapeutic value for detecting and/or altering the expression of Neutrokine-α or Neutrokine-αSV polynucleotides polynucleotide. In addition, polynucleotides spanning the junction of amino acid residues T141 and G142 of the Neutrokine-αSV polypeptide shown in SEQ ID NO: 19 (the V142-K160 amino acid sequence of Neutrokine-α is obviously inserted between them) seem to have diagnostic and therapeutic effects . This T141/G142 spanning polynucleotide presents a higher probability of hybridizing to the Neutrokine-αSV polynucleotide than to the Neutrokine-α polynucleotide. Partial non-limiting non-exclusive examples of such Neutrokine-αSV encoded by polynucleotides of the present invention comprise or consist of amino acid sequences selected from the group consisting of: G121-E163 of SEQ ID NO: 19; E122-E163; G123-E163 ; N124-E163; S125-E163; S126-E163; Q127-E163; N128-E163; S129-E163; R130-E163; -E163; G137-E163; P138-E163; E139-E163; E140-E163; T141-E163; ; W149-E163; L150-E163; L151-E163; S152-E163; F153-E163; K154-E163; R155-E163; G156-E163; S157-E163; A158-E163; -E163; K162-E163; G121-K162; G121-E161; G121-E160; G121-L159; G121-L151; G121-L150; G121-W149; G121-P148; G121-V147; G121-F146; G121-T145; -E139; G121-P138; G121-G137; G121-Q136; G121-V135; G121-A134; ; G121-S126; G121-S125; G121-N124; G121-G123; and G121-E122. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

Vectors and host cells

The present invention also relates to a carrier comprising the isolated DNA molecule of the present invention, with which the recombinant vector is genetically engineered or otherwise engineered to produce the host group vector of the polypeptide of the present invention genetically engineered or otherwise engineered to produce the present invention A host cell for the polypeptide of the invention, and a method for producing Neutrokine-α and/or Neutrokine-αSV polypeptide by recombinant or synthetic methods.

In one embodiment, a polynucleotide of the invention is linked to a vector, such as a cloning or expression vector. A vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral multiplication generally occurs only in complementary host cells. A polynucleotide can be linked to a vector containing a selectable marker for propagation in a host. Introduction of vector constructs into host cells can be performed by techniques known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic liposome-mediated transfection, electroporation, transfection induced, infected or otherwise. These methods are described in many standard laboratory manuals, such as Davis et al., Fundamental Methods of Molecular Biology (1986).

Generally, recombinant expression vectors include an origin of replication and a selectable marker that allows transformation of host cells, such as the ampicillin resistance gene of Escherichia coli and the TRP1 gene of Saccharomyces cerevisiae, and a promoter derived from a highly expressed gene that directs the transcription of downstream structural sequences . Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins. The heterologous structural sequences are assembled in an appropriate state with translation initiation and termination sequences, preferably a leader sequence capable of directly secreting the translated protein into the periplasmic space or into the extracellular medium. Optionally, the heterologous sequence may encode a fusion protein including an N-terminal identifying peptide with desired characteristics, such as stability or ease of purification of the expressed recombinant product.

In one embodiment, the DNA of the present invention is combined with an appropriate heterologous regulatory factor (such as a promoter or enhancer), such as the lambda-phage PL promoter, E. coli Lac, Trp, PhoA and tac promoters, SV40 early And late promoters, and promoters of retroviral LTRs, etc. Other suitable promoters are also known in the art.

As mentioned above, the expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance genes for cultured eukaryotic cells and tetracycline, kanamycin or ampicillin resistance genes for cultured E. coli and other bacteria. Examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells (such as Saccharomyces cerevisiae or Pichia pastoris (ATCC No. 201178)); insect cells Drosophila S2 and Spodoptera Sf 9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropriate media and culture conditions for the above-mentioned host cells are known in the art.

The host cell can be a higher eukaryotic cell, such as a mammalian cell (eg, a human-derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain can be selected that regulates the expression of the inserted gene sequence, or modifies and processes the gene product in the particular manner desired. Expression from certain promoters can be increased in the presence of certain inducers; thus expression of a genetically engineered polypeptide can be controlled. In addition, different host cells have characteristic and specific mechanisms for translational and post-translational processing and modification (eg, phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to enhance the desired modification and processing of exogenously expressed proteins. The selection of appropriate vectors and promoters for expression in host cells is well known in the art, and those skilled in the art also know the construction of expression vectors, the necessary methods for introducing vectors into hosts and expressing them in hosts.

Efficient expression vectors for use in bacteria are constructed by inserting the structural DNA sequence encoding the desired protein, along with appropriate translation initiation and termination signals, into an operable reader with a functional promoter. The vector contains one or more phenotypic selectable markers and an origin of replication to maintain the vector and, if necessary, provide for amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Bacillus typhimurium, and species within the genera Pseudomonas, Streptomyces and Staphylococcus, although other species may alternatively be used. Expression vectors for use in cells typically, but not limited to, may contain a selectable marker and a bacterial origin of replication derived from a commercially available plasmid containing the genetic elements of the well known cloning vector pBR 322 (ATCC 37017). Such commercially available vectors include, for example, pKK 223-3 (Pharmacia Fine Chemicds, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" portions are combined with the appropriate promoter and structural sequences to be expressed. Preferred carriers for bacteria include pHE4-5 (ATCC NO.209311; and variants thereof), pQE70, pQE60, and pQE-9 available from QIAGEN; pBS vectors available from Stratagene, Phagescript vectors, Bluescript vectors, pNH8A , pNH16A, pNH18A, pNH46A; ptrc 99a, pKK223-3, pKK233-3, pDR540, pRIT5 purchased from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZα, pPIC 9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, pPIC9K , and PAO815 (both available from Invitrogen, Carlsbad, CA). Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmaeia. Other suitable vectors are also known to those skilled in the art.

After transformation of an appropriate host strain and growth to an appropriate cell density, the selected promoter is induced by an appropriate method (eg temperature shift or chemical induction) and the cells are cultured for an additional period of time. Cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells used for protein expression can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, which methods are well known to those skilled in the art.

In one of the above embodiments, Pichia pastoris is used to express Neutrokine-alpha protein in a eukaryotic cell system. Pichia pastoris is a methylotrophic yeast that metabolizes methanol as its sole carbon source. A major step in the methanol metabolic pathway is the oxidation _of methanol with O to formaldehyde. This reaction is catalyzed by alcohol oxidase. To metabolize methanol as its sole carbon source, Pichia pastoris must produce high levels of alcohol oxidase because alcohol oxidase has a low affinity for _O2 . Therefore, the promoter regions of one or both alcohol oxidase genes (AOX 1 ) were highly active in the growth medium with methanol as the main carbon source. In the presence of methanol, the alcohol oxidase enzyme produced from the AOX 1 gene comprises about 30% or more of the total soluble protein in Pichia pastoris. See Ellis, SB, et al., Cell Mol. Biol. 5:1111-21 (1985); Koutz, PJ, et al., Yeast 5:167-77 (1989); Tschopp, JF, et al., Nucleic Acids Res. 15:3859-76 (1987). Thus, heterologous coding sequences, such as Neutrokine-alpha or Neutrokine-alphaSV polypeptides of the invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence, produce unusually high levels in Pichia pastoris grown in the presence of methanol. Express.

In one embodiment, the plasmid vector pPIC9K is used to express DNA encoding Neutrokine-α or Neutrokine-αSV polypeptides in a Pichia yeast system substantially as described in "Pichia Protocol: Molecular Biology." Methods", D.R. Higgins and J. Cregg, eds., Humana Press, Totowa, NJ, 1998. This expression vector makes the Neutrokine-α or Neutrokine-αSV protein expression and secretion.

Those skilled in the art realize that many other yeast vectors can be used to replace pPIC9K, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZα, pPIC 9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, and PAO815, as long as the proposed expression construct provides the appropriate localization signals for transcription, translation, secretion (if desired), etc., including the required in-frame AUG.

In one embodiment, high-level expression of heterologous coding sequences such as Neutrokine-α or Neutrokine-αSV polynucleotides of the present invention can be achieved by cloning the heterologous polynucleotides of the present invention into expression vectors such as pGAPZ or pGAPZα, It is obtained by cultivating yeast in the absence of methanol.

Transcription of the DNA encoding the polypeptide of the present invention by higher eukaryotes is enhanced by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually 10-300 bp in length, that act on a promoter to increase its transcription. Examples include the SV40 enhancer 100-270 bp on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma virus enhancer on the late side of the replication origin, and the adenovirus enhancer.

Various mammalian cell culture systems can also be used to express recombinant proteins. Mammalian expression lines include, for example, the COS-7 cell line of monkey kidney fibroblasts described by Gluzman (Cell 23:175 (1981)), and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO , Hela and BHK cell lines. Mammalian expression vectors contain an origin of replication, an appropriate promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'Flanking untranscribed sequences. DNA sequences derived from the SV40 spliceosome, and polyadenylation sites can also be used to provide the desired untranscribed genetic elements.

In a specific embodiment, constructs expressing a portion of the extracellular domain of Neutrokine-alpha (e.g. amino acid residues Ala 134-Leu285) are preferred. Those skilled in the art can use the polynucleotide and polypeptide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19, respectively, to design polynucleotide primers to produce this expression construct.

In another embodiment, constructs expressing the entire putative extracellular domain of Neutrokine-alpha (ie Gln73-Leu285 amino acid residues) are preferred. Those skilled in the art can use the polynucleotide and polypeptide sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19, respectively, to design polynucleotide primers to generate such expression constructs body.

In addition to encompassing host cells containing said vector constructs, the present invention also encompasses primary, secondary, and immortalized host cells of vertebrate, especially mammalian, origin that have been engineered to delete or Replace endogenous genetic material (such as Neutrokine-α coding sequence), and/or include genetic material (such as heterologous polynucleotide sequence), which is related to, activates, alters the Neutrokine-α polynucleotide of the present invention and/or amplifying Neutrokine-alpha polynucleotides. For example, methods known in the art can be used to operably combine heterologous control regions (such as promoters and/or enhancers) and endogenous Neutrokine-alpha polynucleotide sequences by homologous combination (see for example June 24, 1997 U.S. Patent No. 5,641,670 issued on September 26, 1996; International Publication No. WO 96/29411 issued on September 26, 1996; International Publication No. WO 94/12650 issued on August 4, 1994; Koller et al., American Academy of Sciences Acta Proc. 86:8932-8935 (1989); and Zijtstra et al., Nature 342:435-438 (1989), incorporated by reference in their entireties as indicated).

The aforementioned host cells can be used in a conventional manner to produce gene products encoded by recombinant sequences. Alternatively, cell-free translation systems can also be used to produce polypeptides of the invention using RNA derived from the DNA constructs of the invention.

Polypeptides of the invention may be expressed or synthesized in modified forms, such as fusion proteins (including polypeptides bound by peptide bonds to heterologous protein sequences of (different proteins)), and may include not only secretion signals, but also additional heterologous functional domains . Such a fusion protein can be obtained by linking the polynucleotide of the present invention and a desired nucleic acid sequence encoding a desired amino acid sequence to each other in an appropriate reading frame, and expressing the fusion protein product by methods known in the art. Alternatively, such fusion proteins can be produced by protein synthesis methods, such as using a peptide synthesizer. Thus, for example, regions of additional amino acids, especially polar amino acids, may be added to the N-terminus of a polypeptide to improve stability and persistence during purification, or during subsequent holding and storage. Likewise, peptide moieties can be added to polypeptides to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. Adding peptide components to polypeptides to produce secretion or excretion, or to improve stability and promote purification, etc., is a well-known and commonly used method in the art.

In one embodiment, polynucleotides encoding Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention may be fused to the pelB pectate lyase signal sequence to increase the expression of this polypeptide in Gram-negative bacteria. Expression and purification efficiency. See US Patent Nos. 5,576,195 and 5,846,818, which are incorporated by reference in their entirety.

A preferred fusion protein comprises a heterologous region of an immunoglobulin, which is used to stabilize and purify the protein. For example EP-A-0464533 (Canadian counterpart application 2045869) discloses fusion proteins comprising parts of the constant regions of immunoglobulin molecules and other human proteins or parts thereof. In many cases, the Fc part in fusion proteins is very advantageous for therapeutic and diagnostic purposes, thus for example improving pharmacokinetic properties (EP-A 0232262). On the other hand, it is necessary to be able to remove this Fc portion after the fusion protein has been expressed, detected and purified in the advantageous manner described above. This is the case when the Fc part becomes a hindrance in therapy and diagnosis, eg when the fusion protein is used as an antigen for immunization. In drug development, for example, human proteins such as hIL-5 have been fused to the Fc portion for high-throughput screening assays to identify antagonists of hIL-5. See D. Bennett et al., J. Molecular Rec. 8:52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

The polypeptides of the present invention include naturally purified products, chemically synthesized products, and products produced by recombinant methods from prokaryotic or eukaryotic hosts, such as bacteria, yeast, higher plants, insects and mammalian cells. Depending on the host used in the recombinant production method, the polypeptides of the invention may be glycosylated or non-glycosylated. In addition, polypeptides of the invention may also include initially modified methionine residues resulting from host-mediated processing.

Polypeptides of the present invention can be chemically synthesized using techniques known in the art (for example: Creighton, 1983, Principles of Protein Structure and Molecules, W.H.Freeman & Co., N.y., and Hunkapiller, M., et al., 1984, Nature 310: 105-111 ). For example, peptides corresponding to fragments of intact Neutrokine-alpha or Neutrokine-alpha SV polypeptides of the invention can be synthesized using a peptide synthesizer. In addition, if desired, non-canonical amino acids or chemical amino acid analogs can be introduced as substitutions or additions to the Neutrokine-alpha or Neutrokine-alphaSV polynucleotide sequence. Non-classical amino acids include but are not limited to D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline acid, n-citrulline, cystine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluorinated amino acids, designer amino acids such as b -Methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and general analogs of amino acids. In addition, amino acids can be D (dextrorotatory) or L (left-rotatory).

The invention encompasses Neutrokine-α or Neutrokine-αSV polypeptides specifically modified during or after translation, for example by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteases cutting, linking with antibody molecules or other cellular ligands, etc. Any chemical modification can be performed by known methods, including but not limited to chemical cleavage by cyanogen bromide, trypsin, protease, papain, V8 protease, _NaBH4 , acetylation, formylation, oxidation, reduction, in the presence of coating In the case of mycin, it is metabolized and synthesized.

Other post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of the N- or C-terminus, attachment of chemical components to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains , and addition or deletion of N-terminal methionine residues due to prokaryotic host cell expression. Polypeptides can also be modified with detectable labels, such as enzymatic labels, fluorescein labels, isotopic or affinity labels that detect and separate proteins. Additionally, polypeptides of the invention may be modified by iodination.

In one embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention may also be labeled with biotin. In another related embodiment, biotinylated Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention, for example, can be used to identify one or more Neutrokine-α and/or Neutrokine-αSV receptors or other common Imaging agents or tools for receptor or co-ligand molecules.

The present invention also provides chemically modified derivatives of Neutrokine-α or Neutrokine-αSV, which have additional advantages such as increased solubility, stability and in vivo or extracorporeal circulation time of the polypeptide, or reduced immunogenicity (see U.S. Patent No. 4179337). The chemical components used for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, etc. Polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule, and may include 1, 2, 3, or more attached chemical moieties.

The polymers can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1-100 KDa (the term "about" means that in the preparation of polyethylene glycol, some molecules are heavier or lighter than the standard molecular weight) for ease of use and production. Depending on the desired therapeutic principles (e.g., duration of sustained release desired, effect on biological activity, ease of application, degree or loss of antigenicity, and known effects of polyethylene glycol on therapeutic proteins or analogs), may be Use other sizes of polyethylene glycol. For example polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 , 10,000, 10,500, 11,000, 11,500, 12000, 12500, 13500, 14500, 1500, 15500, 16500, 17500, 17500, 18500, 19500, 25000, 30000, 35000, 40000, 40000,40000,40000,40000,40000 , 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 100,000 KDa.

As indicated above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, by U.S. Patent No. 5,643,575; Morpurgo et al., Biochem. Biotechnology Appl. 56:59-72 (1996); Vorobjev et al., Nucleoside Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjugate Chemistry 10:638-646 (1996), which is incorporated by reference in its entirety.

When polyethylene glycol molecules (or other chemical components) are attached to proteins, their effects on protein functional domains or antigenic domains should be considered. A number of attachment methods are known in the art, such as EPO 401384 (coupling of PEG to G-CSF), also described in Malik et al., Exp. Hematol. 20:1028-1035 (1992) (pegylation of GM-CSF with tresyl chloride) . For example, polyethylene glycol can be covalently bound to amino acid residues through reactive groups such as free amino or carboxyl groups. Reactive groups are those groups to which activated polyethylene glycol molecules can bind. Amino acid residues with free amino groups may, for example, include lysine residues and N-terminal amino acid residues; amino acid residues with free carboxyl groups may include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Thiol groups can also be used as reactive groups for attaching polyethylene glycol molecules. Attachment at an amino group is preferred for therapeutic purposes, such as at the N-terminus or a lysine group.

As indicated above, polyethylene glycol can be attached to proteins by linking with any amino acid. For example, polyethylene glycol can be attached to proteins through covalent bonds to lysine, histamine, aspartic acid, glutamic acid or cysteine. One or more reactive chemistries are available for the attachment of PEG to specific amino acid residues (such as lysine, histamine, aspartic acid, glutamic acid, or cysteine) in proteins, or to one of the protein's More than one amino acid residue (such as lysine, histamine, aspartic acid, glutamic acid or cysteine and combinations thereof)

One may specifically desire proteins that are chemically modified at the N-terminus. Taking polyethylene glycol as an example, various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules and protein (or peptide) molecules in the reaction mixture can be selected for pegylation Type of reaction, method to obtain N-terminal PEGylated proteins of choice. A method of obtaining an N-terminally PEGylated preparation (ie, if it is desired to separate this fraction from other mono-PEGylated fractions) may be by purifying the N-terminally PEGylated material from the population of PEGylated protein molecules. Chemically modified selected proteins modified at the N-terminus can be achieved by a reductive alkylation reaction that exploits the different reactions of different types of primary amino groups (relative to the N-terminal lysine) suitable for derivatization in a particular protein sex. Under appropriate reaction conditions, selective derivatization of the protein at the N-terminus with the carbonyl group containing the polymer is substantially achieved.

As indicated above, PEGylation of proteins of the invention can be achieved by a number of methods. For example, polyethylene glycol can be attached to the protein directly or through an intervening linker. Linker systems for attaching polyethylene glycol to proteins are found in Delgado et al., Crit. Rev. Thera. Drug CarrierSys, 9: 249-304 (1992); Francis et al., Intern. J of Hematol. 68: 1-18 (1998) ; US Patent No. 4,002,531; US Patent No. 5,349,052; WO95/06058; and WO98/32466. The above documents are incorporated by reference in their entirety.

A system for attaching polyethylene glycol directly to amino acid residues in proteins without an intervening linker utilizes tresylated MPEG with 2,2,2-trifluoroethanesulfonyl chloride (ClSO ₂ CH ₂ CF ₃ ) to modify monomethoxy polyethylene glycol (MPEG). Based on the reaction of proteins with tresylated MPEG, polyethylene glycol is directly attached to the amine groups of proteins. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting a protein of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, US Patent No. 5,612,460 (incorporated by reference in its entirety) discloses urethane linkers linking polyethylene glycol to proteins. Protein-polyethylene glycol conjugates in which polyethylene glycol is attached to the protein via a linker can also be produced by reacting the protein with a compound such as MPEG-succinimidyl succinate, using 1, 1'-carbonyldiimidazole activated MPEG, MPEG-2,4,5-trichlorophenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives. Additional polyethylene glycol derivatives and reaction chemistries for the attachment of polyethylene glycol to proteins are described in WO 98/32466, which is incorporated by reference in its entirety. PEGylated protein products produced using the chemical reaction procedures described herein are included within the present invention.

The number of polyethylene glycol moieties (ie, degree of substitution) attached to each protein of the invention can also vary. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules can be attached to a PEGylated protein of the invention on average. Similarly, the average degree of substitution is between 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11- 13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol components per protein molecule. Methods for determining the degree of substitution are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Neutrokine-α and/or Neutrokine-αSV polypeptides can be recovered and purified by known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") is preferred for purification.

Neutrokine-α polypeptide

The Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention may be monomers or multimers (ie, dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of Neutrokine-[alpha] and Neutrokine-[alpha]SV polypeptides of the invention, their preparation and compositions (preferably pharmaceutical compositions) comprising them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In other embodiments, the multimers of the present invention are at least dimers, trimers or tetramers.

Multimers encompassed by the present invention may be homopolymers or heteropolymers. The term "homologous polymer" as used herein refers to a multimer comprising only Neutrokine-α and/or Neutrokine-αSV polypeptides (including fragments, variants and fusion proteins of Neutrokine-α and/or Neutrokine-αSV) of the present invention. These homologous polymers may contain Neutrokine-α and/or Neutrokine-αSV polypeptides with identical or different amino acid sequences. In a specific embodiment, a homologous polymer of the invention is a multimer comprising only Neutrokine-α and/or Neutrokine-αSV polypeptides having the same amino acid sequence. In another specific embodiment, the homologous polymers of the invention are multimers comprising Neutrokine-α and/or Neutrokine-αSV polypeptides having different amino acid sequences. In specific embodiments, the multimer of the present invention is a homodimer (such as containing Neutrokine-α and/or Neutrokine-αSV polypeptides with the same or different amino acid sequences), or a homotrimer (such as containing Neutrokine-α and/or Neutrokine-αSV polypeptides having identical or different amino acid sequences). In a preferred embodiment, the multimers of the invention are homotrimers. In another embodiment, the homomultimers of the present invention are at least homodimers, homotrimers or homotetramers.

The term "heterologous polymer" as used herein refers to a polymer containing heterologous polypeptides (ie, polypeptides of different proteins) in addition to the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention. In a specific embodiment, the multimers of the invention are heterodimers, heterotrimers or heterotetramers. In other embodiments, the heteropolymers of the present invention are at least heterodimers, heterotrimers or heterotetramers. In another non-exclusive embodiment, the heterologous polymer of the invention comprises a CD40 ligand polypeptide sequence, or a biologically active fragment or variant thereof.

The multimers of the invention may be hydrophobic, hydrophilic, ionic and/or covalently associated and/or may be indirectly linked, for example via liposomes. Thus in one embodiment, a multimer of the invention, such as a homodimer, or a homotrimer, is formed when a polypeptide of the invention is contacted with another polypeptide in solution. In another embodiment, the heterologous polymer of the present invention, such as heterotrimer or heterotetramer, is formed when the polypeptide of the present invention is in solution with the antibody of the polypeptide of the present invention (including the fusion protein of the present invention) Formed upon contact with antibodies against heterologous polypeptide sequences. In other embodiments, multimers of the invention are formed through a covalent relationship with and/or with Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention. This covalent relationship may comprise one or more amino acid residues contained in the polypeptide sequence (such as the polypeptide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 19 or the polypeptide encoded by the deposited cDNA clone related to the present invention the sequence of). In one instance, the covalent association is a cross-link between cysteine residues in the polypeptide sequence that interact with each other in the native (ie, naturally occurring) polypeptide. In another instance, the covalent association is the result of chemical or recombinant manipulation. Alternatively, the covalent relationship may comprise one or more amino acid residues comprised by the heterologous polypeptide sequence in the Neutrokine-α and/or Neutrokine-αSV fusion protein. In one embodiment, the covalent association is between heterologous sequences comprised in a fusion protein of the invention (see, eg, US Patent No. 5,478,925). In a specific embodiment, the covalent association is between heterologous sequences comprised in the Neutrokine-α-Fc and/or Neutrokine-αSV-Fc fusion proteins of the invention. In another specific embodiment, the covalent association of the fusion protein of the invention is between a heterologous polypeptide sequence from another TNF family ligand/receptor member capable of forming Covalently associated polymers, such as oseteoprotegerin (see International Publication No. WO 98/49305, the contents of which are incorporated by reference in their entirety). In another specific embodiment, the covalent association of the fusion protein of the invention is between heterologous polypeptide sequences from CD40L or a soluble fragment thereof. In another embodiment, two or more Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides of the invention are linked by a synthetic linker (eg, a peptide, carbohydrate or soluble polymer linker). Such peptide linkers are described, for example, in US Patent No. 5,073,627 (incorporated by reference). Proteins comprising multiple Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides separated by peptide linkers can be produced using conventional recombinant DNA methods.

Another method for preparing a multimer of Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention comprises the use of Neutrokine-α and/or Neutrokine-αSV polypeptides fused to leucine zipper or isoleucine zipper polypeptide sequences . Leucine zipper or isoleucine zipper domains are polypeptides that promote multimerization of proteins in which the zipper structure is found. Leucine zippers were originally identified in several DNA binding proteins (Landschulz et al., Science 240:1759 (1988)) and have been found in many different proteins. Known leucine zippers or isoleucine zippers are naturally dimerized or trimerized peptides and derivatives thereof. Leucine zippers suitable for producing soluble multimeric Neutrokine-alpha and/or Neutrokine-alpha SV proteins are described, for example, in PCT application WO 94/10308, which is incorporated by reference. Recombinant fusion proteins comprising soluble Neutrokine-α and/or Neutrokine-αSV fused to dimerized or trimerized peptides in solution are expressed in appropriate host cells and extracted from cultures using methods known in the art. The resulting soluble multimerized Neutrokine-α and/or Neutrokine-αSV are recovered in the supernatant.

Some members of the TNF family of proteins are believed to be in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993). Therefore, the trimerized Neutrokine-α and/or Neutrokine-αSV has enhanced biological activity. Preferred leucine zipper components are those that preferentially form trimers. As described in Hoppe et al. (FEBS Communication 344:191, (1994) and U.S. Patent Application Serial No. 08/446922, a leucine zipper derived from pulmonary surfactant protein D (SPD) was disclosed. Others derived from naturally occurring The peptides of the trimeric protein can be used to prepare trimerized Neutrokine-α and/or Neutrokine-αSV.

-Neutrokine-α或Flag

-Neutrokine-αSV融合蛋白中的Flag

多肽序列间的相互作用而联系的。在另一实施方案中，本发明的蛋白是通过包含于Flag

-Neutrokine-α或Flag

-Neutrokine-αSV融合蛋白的异源多肽序列与抗Flag抗体之间的相互作用而缔合的。In another embodiment, the protein of the present invention is contained in Flag

-Neutrokine-α or Flag

-Flag in Neutrokine-αSV fusion protein

Interactions between polypeptide sequences. In another embodiment, the protein of the present invention is contained in Flag

-Neutrokine-α or Flag

-Heterologous polypeptide sequence of Neutrokine-αSV fusion protein and anti-Flag associated with the interaction between antibodies.

The multimers of the invention can be produced using chemistries known in the art. For example, polypeptides to be included in a multimer of the invention can be chemically cross-linked using methods known in the art for optimization of linker molecules and linker molecule lengths (see US Patent No. 5,478,925, incorporated by reference). Alternatively, multimers of the present invention can be produced by methods known in the art by forming one or more molecular Internal crosslinking. (See US Patent No. 5,478,925, incorporated by reference). In addition, polypeptides of the present invention may be routinely modified by adding cysteine or biotin to the C- or N-terminus of the polypeptide, and methods known in the art may be used to generate multimers of polypeptides containing one or more of these modifications ( See US Patent No. 5,478,925, incorporated by reference). Additionally, liposomes containing the polypeptide components desired to be included in the multimers of the invention can be produced by methods known in the art (see US Patent No. 5,478,925, incorporated by reference).

Alternatively, the multimers of the invention can be produced by genetic engineering methods known in the art. In one embodiment, a polypeptide comprised in a multimer of the invention is produced recombinantly using the fusion protein approach described herein or other methods known in the art (see US Patent No. 5,478,925, incorporated by reference). In a specific embodiment, the polynucleotide encoding the homodimer of the present invention is obtained by linking the polynucleotide sequence encoding the polypeptide of the present invention with the sequence encoding the linker polypeptide, and then reverse direction from the C-terminus to the N-terminus of the starting point. (deleted leader sequence) linked to a synthetic polynucleotide encoding the translation product of the polypeptide (see US Patent No. 5478925, incorporated by reference). In another embodiment, recombinant polypeptides of the invention that contain a transmembrane domain and that can be incorporated into liposomes by membrane reconstitution methods are produced using recombinant methods described herein or other methods known in the art (see Incorporation Referenced US Patent No. 5478925).

In one embodiment, the present invention provides an isolated Neutrokine-α polypeptide having the amino acid sequence encoded by the cDNA clone of ATCC deposit number 97768, or having the amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2) , or the present invention provides an isolated polypeptide comprising a part (ie, a fragment) of the above polypeptide. In another embodiment, the present invention provides an isolated Neutrokine-αSV polypeptide having the amino acid sequence encoded by the cDNA clone of ATCC Accession No. 203518, or having the amino acids shown in Figures 5A and 5B (SEQ ID NO: 19) Sequences, or polypeptides comprising parts and (ie, fragments) of the above-mentioned polypeptides are provided.

The polypeptide fragment of the present invention includes a polypeptide comprising or consisting of the amino acid sequence shown in (SEQ ID NO: 2), the amino acid sequence encoded by the cDNA in the plasmid with ATCC deposit number 97768, or the nucleic acid sequence contained in the deposited clone. The nucleotide sequence or the amino acid sequence encoded by the nucleic acid sequence shown in Figures 1A and 1B (SEQ ID NO: 1) is hybridized (such as under stringent hybridization conditions) to the complementary strand.

In addition, the polypeptide fragment of the present invention includes a polypeptide comprising or consisting of the amino acid sequence shown in (SEQ ID NO: 19), the amino acid sequence encoded by the cDNA contained in the plasmid with ATCC deposit number 203518, or the amino acid sequence encoded by the plasmid with the deposited The nucleotide sequence contained in the clone or the amino acid sequence encoded by the nucleic acid sequence complementary to the nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO: 18) hybridizes (eg, under stringent hybridization conditions).

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or consisting of an amino acid sequence encoded by a nucleic acid that hybridizes (eg, under stringent hybridization conditions) to the complementary strand of the nucleotide sequence shown in SEQ ID NO: 21.

The polypeptide fragments of the present invention also include a polypeptide comprising or hybridizing with the amino acid sequence shown in SEQ ID NO: 23, or by a complementary strand to the nucleotide sequence shown in SEQ ID NO: 22 (such as under stringent hybridization conditions ) consists of the amino acid sequence encoded by the nucleic acid.

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or comprising the amino acid sequence shown in SEQ ID NO: 28, or hybridizing with a complementary strand to the nucleotide sequence shown in SEQ ID NO: 27 (such as under stringent hybridization conditions ) consists of the amino acid sequence encoded by the nucleic acid.

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or comprising the amino acid sequence shown in SEQ ID NO: 30, or hybridizing with a complementary strand to the nucleotide sequence shown in SEQ ID NO: 29 (such as under stringent hybridization conditions ) consists of the amino acid sequence encoded by the nucleic acid.

The polypeptide fragments of the present invention include a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence encoded by the cDNA contained in the deposited clone, or the amino acid sequence contained in the deposited clone, or shown in FIG. The amino acid sequence encoded by the nucleic acid shown in 1A and 1B (SEQ ID NO: 1), or the nucleotide sequence of its complementary strand hybridizes (eg, under stringent hybridization conditions). A protein fragment may be "free-standing", or contained within a larger polypeptide, the fragment forming part or a region of the polypeptide, preferably as a single contiguous region. Polypeptide fragments of the present invention typically include or consist of 1-50, 51-100, 101-150, 151-200, 201-250, and/or 251-285 amino acid residues of SEQ ID NO: 2 fragments. Additionally, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.

In a specific embodiment, the polypeptide fragment of the present invention comprises or consists of 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152, 166-181, 185-209, 210-221, 226-237, 244-249, 253-265 and/or 277-284 amino acid residues. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Those skilled in the art will appreciate that mutations directed to a region of the Neutrokine-alpha polypeptides of the invention that encompasses areas not found in the Neutrokine-alpha SV polypeptide sequence can affect the observed biological activity of the Neutrokine-alpha polypeptides. An insert of 19 amino acids (i.e. Val 142-Lys 160 amino acid residues of the sequence shown in Figures 1A and 1B and SEQ ID NO: 2). More particularly, such residues of the Neutrokine-α polypeptide sequence capable of site-directed mutagenesis include, but are not limited to, the following amino acid residues of the Neutrokine-α polypeptide shown in SEQ ID NO: 2: V142; T143; Q144; D145; C146 ; L147; Q148; L149; I 150; A151; D152; S153; E154; T155; P156; T157; I158; Q159 and K160. Polynucleotides encoding Neutrokine-alpha polypeptides with one or more mutations within the V142-K160 region of SEQ ID NO: 2 are contemplated. Polypeptides encoded by these polynucleotides are also encompassed within the scope of the present invention.

Polypeptide fragments may be "free-standing", or contained within a larger polypeptide, the fragment forming a portion or region of the polypeptide, preferably as a single contiguous region. Polypeptide fragments of the present invention typically include, for example, about 1-15, 16-30, 31-46, 47-55, 56-72, 73-104, 105- 163, 163-188, 186-210 and 210-284 amino acid residues. Polypeptide fragments of the present invention further include, for example, about 1-143, 1-150, 47-143, 47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150, 140-200, 140-225 and 140-266 amino acid residues. Further polypeptide fragments may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. Herein, "about" refers to the specific range, and the range of more or less, several, 5, 4, 3, 2, 1 amino acid residues at the amino terminus or carboxy terminus or both. Polynucleotides encoding these polypeptide fragments are also encompassed by the present invention

Other preferred embodiments encompass polypeptide fragments comprising or consisting of the following structures: a putative intracellular domain of Neutrokine-alpha (amino acid residues 1-46 of SEQ ID NO: 2), a putative transmembrane domain of Neutrokine-alpha ( SEQ ID NO: 47-72 amino acid residues of 2), the putative extracellular domain of Neutrokine-α (73-285 amino acid residues of SEQ ID NO: 2), the putative TNF conserved domain of Neutrokine-α (SEQ ID NO: 191-284 amino acid residues of 2), and the puttingative intracellular domain (being the 1-46 amino acid residue fusion of SEQ ID NO: 2) that comprises or is fused in putative extracellular domain by Neutrokine-α A polypeptide consisting of amino acid residues 73-285). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Further preferred embodiments encompass polypeptide fragments comprising or consisting of the putative intracellular domain of Neutrokine-αSV (amino acid residues 1-46 of SEQ ID NO: 19), putative transmembrane domain of Neutrokine-αSV (SEQ ID NO: 47-72 amino acid residues of 19), the putative extracellular domain of Neutrokine-αSV (73-266 amino acid residues of SEQ ID NO: 19), the putative TNF conserved structural domain of Neutrokine-αSV (SEQ ID NO: 172-265 amino acid residues of 19), and the fusion (SEQ ID NO: 1-46 amino acid residues of 19 and 73- 266 amino acid residue fusion) composed of polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Polypeptide fragments encompassed by further embodiments of the invention comprise or consist of the putative beta-sheet regions identified in Figure 7A. These polypeptide fragments of the present invention comprise or consist of Gln144-Ala151, Phe172-Lys173, Ala177-Glu179, Asn183-Ile185, Gly191-Lys204, His210-Val219, Leu226-Pro237, Asn242-Ala251, Gly256-Ile263 and /or Val276-Leu284 amino acid residues. In some other embodiments, these polypeptide fragments of the present invention also comprise or consist of Phe153-Lys154 of SEQID NO: 19, Ala158-Glu160, Asn164-Ile166, Gly172-Lys185, His191-Val200, Leu207-Pro218, Asn223-Ala232 , Gly237-Ile244, and/or Val257-Leu256 amino acid residues, and consist of Phe42-Lys43, Ala47-Glu49, Asn53-Ile55, Gly61-Pro74, His80-Val89, Leu96-Pro107, Asn112 of SEQ ID NO: 23 - Ala121, Gly126-Ile133 and/or Asp146-Leu154 amino acid residues. In other embodiments, these polypeptide fragments of the present invention also include or comprise Gln78-Ala85 of SEQ ID NO: 28, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144-Val153, Leu160-Pro171 , Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu218 amino acid residues, and consist of Gln78-Ala85, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144 of SEQ ID NO:30 -Val153, Leu160-Pro171, Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu218 amino acid residues. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Some polypeptides of the present invention, for example, without limitation, comprise or consist of combinations of amino acid sequences of the present invention, such as [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Lys160] and [Gly161- Gln198] and the fusion of [Val199-Ala248] and [Gly250-Leu285]; [Met 1-Lys113] and [Ile142-Lys160] and [Gly161-Gln198] and [Val199-Ala248] and [ A fusion of Gly250-Leu285]; or [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and [Val199-Ala248] and [Gly250-Leu285] of SEQ ID NO: 2 fusion. Or the fusion of [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Gly161-Gln198] and [Gly250-Leu285] of SEQ ID NO: 2. Other combinations may include polypeptide fragments other than the above combinations (such as the fusion of [Leu114-Thr141] and [Val199-Ala248] and [Gly250-Leu285] and [Ile142-Lys160] of SEQ ID NO: 2). Other combinations may also include heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the present invention (such as [Met1-Lys113] and [Leu114-Thr141] and [Ile142-Lys160] and [Ile142-Lys160] of SEQ ID NO: 2 and Fusion of [Gly161-Gln198] and [Gly250-Leu285] with FLAG tag). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In addition, the polypeptide of the present invention includes or consists of amino acid sequence combinations without limitation, for example, [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [Val180-Ala229] including SEQ ID NO: 19 and the fusion of [Gly230-Leu266]; the fusion of [Met1-Lys113] and [Gly142-Gln179] and [Val180-Ala229] and [Gly230-Leu266] of SEQ ID NO: 19; or the fusion of SEQ ID NO: 19 Fusion of [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [Gly230-Leu266]. Other combinations may include polypeptide fragments other than the above combinations (such as the fusion of [Leu114-Thr141] and [Val180-Ala229] and [Gly230-Leu266] and [Gly142-Gln179] of SEQ ID NO: 19). Other combinations can also include heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the present invention (such as [Met1-Lys113] and [Leu114-Thr141] and [Gly142-Gln179] and [ Gly230-Leu266] fusion with FLAG tag). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Polypeptides of the present invention further, without limitation, for example comprise or consist of amino acid sequence combinations, for example comprising [Met1-Lys106] and [Leu107-Thr134] and [Ile167-Lys184] and [Gly185-Gln224] and [Gly185-Gln224] of SEQ ID NO: 23 and Fusion of [Val225-Ala272] and Gly273-Leu309]; [Met1-Lys106] and [Glu135-Asn165] and [Ile167-Lys184] and [Gly185-Gln224] and [Val225-Ala272] of SEQ ID NO: 23 and The fusion of [Gly273-Leu309], or [Met1-Lys106] and [Leu107-Thr134] and [Glu135-Asn165] and [Ile167-Lys184] and [Gly185-Gln224] and [Gly273-Leu309] of SEQ ID NO: 23 ] fusion. Other combinations may include polypeptide fragments other than the above combinations (such as [Met1-Lys106] and [Gly185-Gln224] and [Ile167-Lys184] and [Val225-Ala272] and [Leu107-Thr134] and [Leu107-Thr134] of SEQ ID NO: 23 and [Gly273-Leu309] fusion). Other combinations may also include heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the present invention (such as [Met1-Lys106] and [Glu135-Asn165] and [Ile167-Lys184] and [ Gly185-Gln224] and [Val225-Ala272] and [Gly273-Leu309] fusions with FLAG tag). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Polypeptides of the present invention additionally, without limitation, for example comprise or consist of amino acid sequence combinations, for example comprising [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [Gly95-Gln132] of SEQ ID NO: 28 and A fusion of [Val133-Ala182] and [Gly183-Ala219]; a fusion of [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182] of SEQ ID NO: 28; or SEQ ID NO: 28 ID NO: fusion of [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182] and [Gly183-Ala219] of 28. Other combinations may include polypeptide fragments other than the above combinations (such as the fusion of [Tyr1-Lys47] and [Gly183-Ala219] and [Val133-Ala182] and [Leu48-Thr75] of SEQ ID NO: 28). Other combinations may also include heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the present invention (such as [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [Gly95-Gln132] of SEQ ID NO: 28 and [Val133-Ala182] fusion with Fc receptor tag). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Polypeptides of the present invention additionally, without limitation, for example comprise or consist of amino acid sequence combinations, for example comprising [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [Gly95-Gln132] of SEQ ID NO: 30 and A fusion of [Val133-Ala182] and [Gly183-Ala219]; a fusion of [Tyr1-Lys47] and [Leu48-Thr75] and [Ile76-Lys94] and [Val133-Ala182] of SEQ ID NO: 30; or SEQ ID NO:30 ID NO: fusion of [Tyr1-Lys47] and [Ile76-Lys94] and [Val133-Ala182] and [Gly183-Ala219] of 30. Other combinations may include polypeptide fragments other than the above combinations (such as the fusion of [Tyr1-Lys47] and [Gly183-Ala219] and [Val133-Ala182] and [Leu48-Thr75] of SEQ ID NO: 30). Other combinations may also include heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the present invention (such as [Leu48-Thr75] and [Ile76-Lys94] and [Gly95-Gln132] and [ Val133-Ala182] and Fc receptor tagged fusion). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Neutrokine-α and/or Neutrokine-αSV polypeptide fragments encompassed by other embodiments of the present invention comprise or consist of functional regions of the polypeptides of the present invention, such as the α region of Garnier-Robaon, the β region, the corner region and the coil region, Chou-Fasman's α region, β region and coiled region, Kyte-Doolittle hydrophilic region and hydrophobic region, Eisenberg's α and β amphipathic region, Karplus-Schulz flexible region, Emini surface formation region and Jameson-Wolf high antigenic index region, as shown in Figure 3 , 6 and Table 1. In a preferred embodiment, the polypeptide fragments of the invention are antigenic. The data shown in columns VIII, IX, XIII and XIV of Table 1 can be used routinely to identify regions of Neutrokine-alpha that exhibit high antigenic potential. Regions of high antigenicity are determined by selecting, from the data shown in columns VIII, IX, XIII and/or IV, values representing regions of the polypeptide that are readily exposed on the surface of the polypeptide under the circumstances in which the antigen is recognized Can occur during the initiation of an immune response. Particularly preferred fragments of the present invention are those comprising regions of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV combining some of the structural features such as some of the above (eg 1, 2, 3 or 4). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the invention provides a polypeptide comprising or consisting of an epitope-bearing portion of a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention. An epitope portion of a polypeptide is an immunogenic or antigenic epitope of a polypeptide of the invention. "Immunogenic epitope" refers to a portion of a protein that elicits an antibody response when the entire protein is an immunogen. On the other hand, "antigenic epitope" refers to a region of a protein molecule to which an antibody can bind. A protein generally has fewer immunogenic epitopes than antigenic epitopes. See, eg, Geysen et al., PNAS 81:3998-4002 (1983).

For selection of polypeptides bearing antigenic epitopes (ie, regions containing regions of protein molecules to which antibodies can bind), relatively short synthetic peptides that mimic part of the protein sequence are well known in the art and generally elicit antisera reactive with the part of the mimic protein. See, eg, Sutcliffe, J.G., Shinnick, T.M., Green, N. and Learrer, R.A. (1983) "Antibodies Reactive to Predetermined Sites on Proteins", Science 219:660-666. Peptides that elicit protein-reactive serum are usually represented in the primary sequence of the protein, can be characterized by some convenient chemical rules, and are defined as neither immunodominant regions of the intact protein (i.e., immunogenic epitopes), nor amino or carboxyl groups end. Peptides or polypeptides of the invention that carry antigenic epitopes are therefore useful for raising antibodies, including monoclonal antibodies that specifically bind to polypeptides of the invention. See, eg, Wilson et al., Cell 37:767-778 (1984), at p777.

The peptide or polypeptide carrying antigenic epitopes of the present invention preferably contains at least 4, at least 5, at least 6, at least 7, preferably at least 8, at least 9, at least 10, at least 11, at least 12, At least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably about 15-30 of the amino acid sequences contained in the polypeptide of the present invention sequence of amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 in length , 95 or 100 amino acid residues. Other preferred antigenic epitopes include the antigenic epitopes disclosed herein and portions thereof.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Neutrokine-α and/or Neutrokine-αSV-specific antibodies include: amino acid residues at positions about Phe115-Leu147 as shown in FIGS. 1A and 1B (SEQ ID NO: 2) A polypeptide composed of bases; comprising or consisting of approximately Ile150-Tyr163 amino acid residues shown in Figures 1A and 1B (SEQ ID NO: 2); comprising or comprising approximately A polypeptide consisting of Ser171-Phe194 amino acid residues; comprising or consisting of Glu223-Tyr246 amino acid residues shown in Figures 1A and 1B (SEQ ID NO: 2); comprising or consisting of Figures 1A and 1B (SEQ ID NO: 2) ) A polypeptide composed of approximately Ser271-Phe278 amino acid residues; "About" herein refers to a specific range, and more or less, a few, 5, 4, 3, 2 or 1 amino acids at the amino-terminal or carboxy-terminal or both ends within this range. These polypeptide fragments have been determined to carry the antigenic epitope of the Neutrokine-α polypeptide by Jameson-Wolf antigenic index analysis, as shown in Figure 3 and Table 1.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Neutrokine-α and/or Neutrokine-αSV-specific antibodies include: amino acid residues at or about Pro32-Leu47 as shown in FIGS. 5A and 5B (SEQ ID NO: 19) Polypeptides composed of bases; polypeptides comprising or consisting of approximately Glu116-Ser143 amino acid residues shown in Figures 5A and 5B (SEQ ID NO: 19); comprising or approximately A polypeptide consisting of Phe153-Tyr173 amino acid residues; comprising or consisting of approximately Pro218-Tyr227 amino acid residues shown in Figures 5A and 5B (SEQ ID NO: 19); comprising or consisting of Figures 5A and 5B (SEQ ID NO: 19) 19) A polypeptide consisting of about Ala232-Gln241 amino acid residues; comprising or consisting of about Ile244-Ala249 amino acid residues shown in Figure 5A and 5B (SEQ ID NO: 19); comprising or consisting of Figure 5A and 5B (SEQ ID NO: 19) shows a polypeptide composed of approximately Ser252-Val257 amino acid residues. Herein, "about" refers to a specific range, and more or less, several, 5, 4, 3, 2 or 1 amino acid residues at the amino-terminal or carboxy-terminal or both ends within this range. Polynucleotides encoding these polypeptides are also encompassed by the present invention. These polypeptide fragments have been determined to carry the antigenic epitopes of the Neutrokine-αSV polypeptide by Jameson-Wolf antigenic index analysis, as shown in Figure 6 and the data in tabular form generated by the Protean component of the DNA*STAR computer program.

The epitope-bearing peptides and polypeptides of the present invention can be produced by any conventional method. See, eg, Houghten, R.A. (1985) A general method for rapid solid-phase synthesis of large numbers of peptides: Specificity of antigen-antibody interactions at the level of individual amino acids, Proc. The method of "Houghten et al. (1986) is described in US Patent No. 4,631,211.

Uses of the epitope-bearing peptides and polypeptides of the invention include, but are not limited to, the induction of antibodies according to methods well known in the art. See, eg, Sutcliffe et al., supra; Wilson et al., supra; Chow, M., et al., PNAS 82: 910-914; and Bittle, F.J., et al., Gen Virus 66: 2347-2354 (1985). Peptides of the invention that carry immunogenic epitopes, ie, portions of proteins that elicit an antibody response when the entire protein is an immunogen, are identified according to methods known in the art. See, eg, Geysen et al., supra. Additionally, U.S. Patent No. 5,194,392 to Geysen describes a method for detecting or determining the sequence of a monomer (amino acid or other compound) that is an epitope that is complementary to a particular paratope (antigen-binding site) of the corresponding antibody The topological equivalent of (ie mimotope). The US patent to Geysen (1989) describes a method for detecting or determining the sequence of a monomer which is a topological equivalent of a ligand complementary to the ligand binding site of a particular receptor. Similarly, Houghten, R.A. et al. (1996) U.S. Patent No. 5,480,971 on peralkylated oligopeptide mixtures disclose linear C1-C7-alkyl-peralkylated oligopeptides and libraries of such peptides, As well as a method of using such an oligopeptide library to determine the sequence of an overalkylated oligopeptide that preferentially binds to a corresponding receptor molecule. Thus, non-peptide analogs of the epitope-bearing peptides of the invention can also be routinely produced by these methods.

The present invention encompasses a polypeptide comprising or consisting of an epitope of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, or a polynucleoside contained in a clone contained in ATCC deposit number 97768 or a polypeptide encoded by a polynucleotide that hybridizes (eg, under the hybridization conditions) to the sequence of SEQ ID NO: 1 or the complement of the cDNA sequence contained in ATCC Accession No. 97768. The present invention also covers a polynucleotide sequence, which comprises or consists of a sequence encoding a polypeptide epitope of the present invention (such as the sequence shown in SEQ ID NO: 1), a polynucleotide of a complementary strand of a polynucleotide sequence encoding an epitope of the present invention acid sequence, and a polynucleotide sequence that hybridizes (under said hybridization conditions) to the complementary strand.

The present invention also encompasses a polypeptide comprising or having an epitope of a polypeptide having an amino acid sequence set forth in SEQ ID NO: 19, or an epitope of a polypeptide sequence encoded by a polynucleotide sequence contained in ATCC Accession No. 203518, or Consists of an epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes (eg, under hybridization conditions) to the complement of the sequence set forth in SEQ ID NO: 18 or to the cDNA sequence contained in ATCC Accession No. 203518. The present invention also covers a polynucleotide sequence, which comprises or consists of an epitope encoding the polypeptide of the present invention (sequence shown in SEQ ID NO: 18), polynucleosides of the complementary strand of the polynucleotide sequence encoding the epitope of the present invention acid sequence, and a polynucleotide sequence that hybridizes (eg, under said hybridization conditions) to the complementary strand.

The term "epitope" as used herein refers to a part of a polypeptide having antigenic or immunogenic activity in animals, preferably mammals, more preferably humans. In a preferred embodiment, the invention encompasses a polypeptide comprising an epitope and polynucleotides encoding such a polypeptide. "Immunogenic epitope" refers to a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by methods for generating antibodies as described above (see, e.g., Geysen et al., Proc. -4002 (1983)). The term "antigenic epitope" refers to a portion of a protein to which an antibody immunospecifically binds its antigen, as determined by any method well known in the art, eg, by the immunoassays described. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. An antigenic epitope need not be immunogenic.

Fragments that are epitopes can be produced by any conventional method (see, e.g., Houghten, PNAS USA 82:5131-5135 (1985), also described in US Patent No. 4,631,211)

In the present invention, antigenic epitopes preferably contain at least 4, at least 5, colored 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least A sequence of 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, most preferably about 15-30 amino acids. Preferably the polypeptide comprising an immunogenic or antigenic epitope is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 in length amino acid residues. Other preferred antigenic epitopes include the antigenic epitopes disclosed herein, and any combination of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (eg, as described by Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used to induce antibodies, e.g., according to methods well known in the art (see, e.g., Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Gene Virus 66: 2347-2354 (1985)). Preferred immunogenic epitopes include those disclosed herein, as well as combinations of 2, 3, 4, 5 or more of these immunogenic epitopes. Polypeptides comprising one or more immunogenic epitopes can be present with a carrier protein, such as albumin, to elicit an antibody response in an animal strain (e.g., rabbit or murine), or if the polypeptide is sufficiently long (at least about 25 amino acids ), this polypeptide can be without carrier. However, immunogenic epitopes comprising as few as 8-10 amino acids have been shown to efficiently generate antibodies capable of binding at least linear epitopes in denatured polypeptides. (as in Western blot).

The epitope-bearing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art, including but not limited to in vivo immunization, in vitro immunization and phage display methods. See, eg, Sutcliffe et al., supra; Wilson et al., supra; and Bittle et al., Gene Virus 66:2347-2354 (1985). If in vivo immunization is used, animals can be vaccinated with free peptide; however, anti-peptide antibody titers can be boosted by conjugating the peptide to a macromolecular carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxin . For example, peptides containing cysteine residues can be coupled to a carrier using a linker such as m-maleimidebenzoic acid-N-hydroxysuccinimide ester (MBS), while other peptides can be coupled to a carrier using more general linking agents such as Glutaraldehyde is coupled to the carrier. Animals such as rabbits, rats and mice are immunized with free peptide or peptide conjugated to a carrier, for example by intraperitoneal and/or intradermal injection of an emulsion containing approximately 100 mg of peptide or carrier protein and Freud 's adjuvant or any other adjuvant known to stimulate an immune response. Several booster injections, for example every about two weeks, are required to provide effective titers of anti-peptide antibodies detectable, for example, by ELISA assay using free peptide adsorbed to a solid surface. Anti-peptide antibody titers in sera of immunized animals can be increased by selecting for anti-peptide antibodies, for example, by adsorbing the peptide to a solid support and eluting the selected antibodies according to methods well known in the art.

Those skilled in the art will recognize and as noted above, polypeptides of the invention comprising immunogenic or antigenic epitopes may be fused to other polypeptide sequences. For example, a polypeptide of the present invention can be fused to a constant region of an immunoglobulin (IgA, IgE, IgG, IgM), or a portion thereof (CH1, CH2, CH3, or any combination thereof and a portion thereof) to generate a chimeric polypeptide. This fusion protein is easy to purify and has an increased half-life in vivo. Chimeric proteins have been shown to consist of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the mammalian heavy or light chains. See, eg, Ep 394827; Tyaunecker et al., Nature, 331:84-86 (1988). Antigen delivery across the epithelial barrier to the immune system is enhanced. Antigens such as insulin have been shown to be conjugated to FcRn binding partners such as IgG or Fc fragments (see eg PCT Publications WO96/22024 and WO99/04813). IgG fusion proteins having a disulfide-linked dimer structure due to desulfurization of the IgG portion have also been found to have a more efficient ability to bind and neutralize other molecules than monomeric polypeptides or fragments thereof. See, eg, Fountoulakis et al., J. Biol. Chem. 270:3958-3964 (1995). The nucleic acid encoding the above-mentioned epitope can also be recombined with the corresponding gene as an epitope tag (such as hemagglutinin (HA) tag or flag tag), so as to facilitate the detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. facilitates the purification of undenatured fusion proteins expressed in human cell lines (Janknecht et al., Proc. Acad. Sci. USA 88:89072-897). In this system, the corresponding gene is subcloned into a vaccinia recombinant plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. This tag serves as the matrix-binding domain of the fusion protein. Extracts from cells infected with recombinant vaccinia virus were applied to a Ni ²⁺ nitriloacetic acid agarose chromatography column, and histidine-tagged proteins could be selectively eluted with a buffer containing imidazole.

In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention and epitope-bearing fragments thereof are fused to heterologous antigens (such as polypeptides, carbohydrates, phospholipids or nucleic acids). In specific embodiments, the heterologous antigen is an immunogen.

In a more specific embodiment, the heterologous antigen is the gp120 protein of HIV or a fragment thereof. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide of the present invention and its epitope-carrying fragments are compatible with the polypeptide sequence of another TNF ligand family member (or its biologically active fragment or its variant ) fusion. In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention are fused to a CD40L polypeptide sequence. In preferred embodiments, the CD40L polypeptide sequence is soluble.

Methods of gene shuffling, motif shuffling, exon shuffling and/or codon shuffling (collectively "DNA shuffling") that can be used to modulate the activity of Neutrokine-α and/or Neutrokine-αSV to efficiently produce Neutrokine-α And/or agonists and antagonists of Neutrokine-αSV. See US Pat. (1998); Hansson, L.O, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechnology 24(2):308-13 (1998), the Literature is incorporated by reference. In one embodiment, alterations to Neutrokine-alpha and or Neutrokine-alpha SV polynucleotides and corresponding polypeptides can be made by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired Neutrokine-α and/or Neutrokine-αSV molecule by homology or site-specific recombination. In another embodiment, Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polynucleotides and corresponding polypeptides may be altered by random mutagenesis by error-prone polymerase chain reaction, random insertion of nucleotides, or other methods prior to recombination. In another embodiment, one or more components, motifs, segments, parts, domains, fragments, etc. of Neutrokine-α and/or Neutrokine-αSV may be combined with one or more components of one or more heterologous molecules. Recombination of individual components, motifs, segments, parts, domains, fragments, etc. In a preferred embodiment, the heterologous molecule is for example TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (in the heterotrimeric LT-α2-β complex found in), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (International Publication No. WO96/14328), AIM-I (International Publication WO97/33899), AIM-II (International Publication WO97/34911), APRIL (Journal of Experimental Methods 188(6):1185-1190), endokine-α (International Publication WO98/07880), OPG, OX40, and nerve growth factor (NGF), and soluble forms Fas, CD 30, CD27, CD40 and 4-IBB, TR2 (International Publication WO96/34095), DR3 (International Publication WO97/33904), DR4 (International Publication WO98/32856), TR5 (International Publication WO98/30693), TR6 (International Publication WO98/30694), TR7 (International Publication WO98/41629), TRANK, TR9 International Publication WO98/56892), TR10 International Publication WO98/54202), 312C2 (International Publication WO98/06842), TR12, CDA and V -FLIP. In other embodiments, the heterologous molecule is any member of the TNF family.

In preferred embodiments, Neutrokine-α and/or Neutrokine-αSV polypeptides (including biologically active fragments or variants thereof) of the invention are fused to soluble CD40L polypeptides or biologically active fragments or variants thereof.

To improve or alter the characteristics of Neutrokine-α and/or Neutrokine-αSV polypeptides, protein engineering can be performed. Recombinant DNA methods known to those skilled in the art can be used to generate new mutant proteins or "muteins" which include one or more amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or increased stability. In addition, they can be purified in high yields and have better stability than the corresponding native polypeptides, at least under certain purification and storage conditions. For example, for many proteins, including the extracellular domains of mature forms of secreted proteins, it is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus without substantial loss of biological function. For example, Ron et al., J. Biol. Chem., 268: 2984-2988 (1993) reported modified KGF proteins that had heparin-binding activity despite loss of 3, 8, or 27 amino-terminal amino acids.

Since the protein of the present invention is a member of the TNF polypeptide family, the N-terminal amino acids of Fig. 1A and 1B (SEQ ID NO: 2) can still retain some biological activities such as stimulating lymphocytes (such as the deletion of the Gly (G) residue at position 191) B cells) proliferate, differentiate and/or activate, and have the ability to be cytotoxic to appropriate target cells. Polypeptides with a further N-terminal deletion including a Gly (G) residue are not expected to retain biological activity, since this residue in TNF-related polypeptides is known to be the start of a conserved domain required for biological activity. However, even if one or more amino acids are deleted from the N-terminus of the protein and one or more functions of the protein are lost, other functional activities can still be retained. Thus, the ability of the shortened protein to induce and/or bind antibodies that recognize the intact protein or its ectodomain may be preserved when a small number of residues of the intact protein or its ectodomain are removed from the N-terminus. Whether particular polypeptides lacking N-terminal residues of the intact protein retain such immunological activity can be determined by routine methods described herein and others known in the art.

Therefore, the present invention further provides a polypeptide and a polynucleotide encoding such a polypeptide, the polypeptide has a deletion from the amino terminal of the amino acid sequence of Neutrokine-α shown in Figures 1A and 1B (SEQ ID NO: 2) until position 191 One or more residues of glycine residues (Gly191 at the amino terminus). In particular, the polypeptide provided by the present invention comprises or consists of the amino acid sequence of residues n ^1-285 of SEQ ID NO: 2, wherein n ¹ is the amino acid sequence of residues 2-190 of the amino acid sequence shown in SEQ ID NO: 2 an integer of . Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, the polynucleotide encoding the polypeptide provided by the present invention comprises or consists of an amino acid sequence selected from the following group: 2-285, 3-285, 4-285, 5-285, 6 of SEQ ID NO: 2 -285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285 , 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31 -285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285 , 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56 -285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285 , 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77-285, 78-285, 79-285, 80-285, 81 -285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285 , 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285, 102-285, 103-285, 104-285, 105-285, 106 -285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-285 , 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-285, 128-285, 129-285, 130-285, 131 -285, 132-285, 133-285, 134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285 , 144-285, 145-285, 146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-285, 155-285, 156 -285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285 , 169-285, 170-285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181 -285, 182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and 190-285. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 92%, 95%, 96% of the polynucleotide sequence encoding Neutrokine-α and/or Neutrokine-αSV polypeptide %, 97%, 98% or 99% identical polynucleotide sequence composition. The invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acids and/or polynucleotides are also encompassed in the present invention, and these polypeptides comprise or consist of at least 80%, 85%, 90%, 92%, 95%, 96%, 97% of the above-mentioned amino acid sequences %, 98% or 99% identical amino acid sequences, and polynucleotides encoding such polypeptides are also included in the present invention.

In addition, since the putative ectodomain of the Neutrokine-α polypeptide of the present invention can stimulate biological activity by itself, the N-terminal and C-terminal amino acid residues of the putative ectodomain of the polypeptide (spanning amino acids Gln73-Leu285 of SEQ ID NO: 2) are still missing. Some biological activity may be retained, such as ligand binding, stimulation of lymphocyte proliferation, differentiation and/or activation, and modulation of cell replication or modulation of target cell activity. However, even if the deletion of one or more amino acids from the N-terminus of the putative ectodomain of a Neutrokine-alpha polypeptide results in the loss of one or more biological functions of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind antibodies that recognize the intact polypeptide or its extracellular domain may be retained when a small number of residues of the intact or mature polypeptide or its extracellular domain are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residues of the complete polypeptide retains such immunological activity can be determined by routine methods described herein and by other methods known in the art.

Therefore, the present invention also provides a polypeptide and a polynucleotide encoding the polypeptide, the polypeptide has one or more deletions from the amino terminal of the Neutrokine-α amino acid sequence shown in SEQ ID NO: 2 up to the 280th glycine residue Residues. In particular, the polypeptide provided by the present invention comprises or consists of the n ² -285 amino acid sequence of SEQ ID NO: 2, wherein n ² is an integer number of amino acid positions 73-280 of SEQ ID NO: 2, 73 position is the first residue position N-terminal to the putative ectodomain of the Neutrokine-alpha polypeptide (SEQ ID NO: 2). Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, in certain embodiments, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: Q-73 to L-285 of SEQ ID NO: 2; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; 80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; L-283; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; 285; A93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K- 99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-235; L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; 285; L-112 to L-285; K-113 to L-285; 1-114 to L-285; F-115 to L-285; EI16 to L-285; 118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-235; E-122 to L-285; G-123 to L-285; L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; 285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; 143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-280; D-152 to L-285; S-153 to L-285; E-154 to L-285; 285; P-156 to L-285; T-157 to L-285; 1-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; 168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-235; L-178 to L-285; E-179 to L-285; 285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; 193 to L-285; F-194 to L-280; I-195 to L-285; Y196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L- 285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-203 to L-285; G-209 to L-285; H-210 to L-285; 212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; 285; S-225 to L-285; L226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; 231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-235; N-235 to L-285; M-236 to L-285; L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L- 285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; 1-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; 256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; 285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 92%, 95% of the polynucleotide sequence encoding the above-mentioned Neutrokine-α and/or Neutrokine-αSV polypeptide, Composition of polynucleotide sequences that are 96%, 97%, 98% or 99% identical. The invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acids and/or polynucleotides are also encompassed by the present invention, and these polypeptides comprise or consist of at least 80%, 85%, 90%, 92%, 95%, 96%, 97% of the above amino acid sequences %, 98% or 99% identical amino acid sequence composition, polynucleotides encoding such polypeptides are also included in the present invention.

Particularly preferred embodiments of the present invention relate to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence that encodes the polynucleotide as shown in Figures 1A and 1B (SEQ ID NO: 2 ) has at least 95%, 96%, 97%, 98%, 99% or 100% identity to the polynucleotide sequence of the Neutrokine-α polypeptide of the amino acid sequence 134-285 shown in ). Preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence whose sequence and code are shown in Figures 1A and 1B (SEQ ID NO: 2) The polynucleotide sequence of the Neutrokine-α polypeptide of the shown 134-285 amino acid sequence has at least 90% identity. More preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence that encodes the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 2). - The polynucleotide sequence of the Neutrokine-α polypeptide at amino acid sequence 285 has at least 95% identity. More preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence that encodes the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 2). - The polynucleotide sequence of the Neutrokine-α polypeptide at amino acid sequence 285 has at least 96% identity.

In addition, more preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence that is identical to the coding sequence shown in Figures 1A and 1B (SEQ ID NO: 2). The polynucleotide sequences of the Neutrokine-α polypeptides showing the 134-285 amino acid sequence have at least 97% identity. In addition, more preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence that is identical to the coding sequence shown in Figures 1A and 1B (SEQ ID NO: 2). The polynucleotide sequences of the Neutrokine-α polypeptides showing the 134-285 amino acid sequence have at least 98% identity. In addition, more preferred embodiments of the present invention relate to nucleic acid molecules comprising or consisting of polynucleotides having a nucleotide sequence that is identical to the coding sequence shown in Figures 1A and 1B (SEQ ID NO: 2). The polynucleotide sequences of the Neutrokine-α polypeptides showing the 134-285 amino acid sequence have at least 99% identity.

In specific embodiments, a polypeptide consisting of one of the following N-terminal deletion polypeptide fragments of Neutrokine-α and/or Neutrokine-αSV is preferred: Ala71-Leu285 amino acid residues of SEQ ID NO: 2, Ala81-Leu285 Amino acid residues, Leu112-Leu285 amino acid residues, Ala134-Leu285 amino acid residues, Leu147-Leu285 amino acid residues, and Gly161-Leu285 amino acid residues, polynucleotides encoding these polypeptides are also included in the present invention .

Similarly, many biologically functional C-terminal deletion muteins are known. For example, interferon gamma improves 10-fold activity by missing 8-10 amino acid residues at the carboxy-terminal of its protein (Dobeli et al., Journal of Biotechnology 7:199-216 (1988)). Since the protein of the present invention is a member of the TNF polypeptide family Member, the C-terminal amino acid until the deletion of the 284-position leucine residue is expected to retain most of the biological activities, such as ligand binding, ability to stimulate lymphocyte (such as B cell) proliferation, differentiation and/or activation, regulation of cell replication ability. Polypeptides deleted up to about 10 additional C-terminal or residues (i.e. up to glycine residue 274) may also retain some activity, such as receptor binding activity, although such polypeptides lose a portion extending to Leu284 of SEQ ID NO:2 The conserved TNF domain. However, even if the deletion of one or more amino acids from the C-terminus of a protein results in the loss of one or more biological functions of the protein, other functions may still be retained. Thus, when a small number of residues of the intact or mature protein are removed from the C-terminus, the ability of the shortened protein to induce and/or bind antibodies that recognize the intact or mature protein remains. Whether a particular polypeptide lacking the C-terminal residues of the intact protein retains such immunological activity can be determined by routine methods described herein and other methods known in the art.

Therefore, the present invention further provides a polypeptide and a polynucleotide encoding the polypeptide, the polypeptide has a deletion from the carboxyl terminal of the amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2) to glycine 274 (Gly274) one or more residues. In particular, the present invention provides a polypeptide comprising or consisting of the amino acid sequence of residues ¹ -m1 of the amino acid sequence of SEQ ID NO:2, wherein ^m1 is one of the amino acid residues 274-284 of SEQ ID NO:2 any integer in between. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of 1-274, 1-275, 1-276, 1- 277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283, and 1-284 residues. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The present invention also relates to a nucleic acid molecule comprising or consisting of at least 80%, 85%, 90%, 92%, 95%, 96% of a polynucleotide sequence encoding Neutrokine-α and/or Neutrokine-αSV polypeptide, Composition of polynucleotide sequences that are 97%, 98% or 99% identical. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides comprise or consist of at least 80%, 85%, 90%, 92%, 95%, 96%, Amino acid sequence compositions with 97%, 98% or 99% identity, and polynucleotides encoding these polypeptides are also encompassed by the present invention.

The present invention also provides a polypeptide comprising or consisting of one or more amino acids deleted from the amino-terminus and carboxy-terminus, which can be described as having n ¹ -m ¹ residues of SEQ ID NO: 2, wherein n ¹ and m ¹ is an integer as described above. The present invention also includes nucleotide sequences encoding polypeptides comprising or consisting of a portion of the complete Neutrokine-alpha amino acid sequence encoded by the deposited cDNA clone of ATCC Accession No. 97768, wherein the portion does not include the complete amino acid sequence (or these N - and C-terminal deletions) amino acids 1-190 at the N-terminus, or amino acids 1-11 at the C-terminus. Polynucleotides encoding all of the aforementioned deletion polypeptides are also encompassed by the present invention.

Similarly, deletions from the C-terminal residues of the putative ectodomain of Neutrokine-α up to the 79-position leucine residue of SEQ ID NO: 2 may retain some biological activities, such as ligand binding, stimulation of lymphocytes (such as B cells) Proliferation, differentiation and/or activation, and regulation of cell replication or modulation of target cell activity. Polypeptides with further C-terminal deletions including Leu79 of SEQ ID NO: 2 are not expected to retain biological activity.

However, even if one or more amino acids are deleted from the C-terminus of a polypeptide resulting in the loss of one or more biological functions of the polypeptide, other biological activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind antibodies that recognize the full, mature polypeptide or its extracellular domain may be retained when a small number of residues of the full, mature polypeptide or its extracellular domain are removed from the C-terminus. Whether a particular polypeptide lacking a putative C-terminal residue of the extracellular domain retains such immunological activity can be determined by routine methods described herein and by other methods known in the art.

Therefore, the art also provides a polypeptide and a polynucleotide sequence encoding the polypeptide, the polypeptide has a deletion from the amino acid sequence carboxy-terminal of the putative ectodomain of the Neutrokine-α polypeptide shown in SEQ ID NO: 2 up to the 79th leucine one or more residues. In particular, the present invention provides a polypeptide comprising or consisting of an amino acid sequence consisting of 73- ^m2 residues of the amino acid sequence shown in SEQ ID NO:2, wherein ^m2 is the 79-m2 residue of the amino acid sequence shown in SEQ ID NO:2 Of the 285 amino acid residue positions - an integer position, residue 78 is the first residue at the C-terminus of the putative extracellular domain of the Neutrokine-α polypeptide (SEQ ID NO: 2). Polypeptides encoded by these polynucleotides are also encompassed by the present invention. More particularly, in certain embodiments the invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: Q-73 to Leu-285 of SEQ ID NO: 2; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F -278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272 Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; -73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L -253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to S-247 Q-73 to Y-246; Q-73 to C-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; -73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T -228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222 Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q -73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q73 to H-210; Q-73 to G -209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203 Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; -73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L186; Q-73 to I-185; Q-73 to K-184 Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; -73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F -165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159 Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; -73 to D-152; Q-73 to A-151; Q-73 to 1-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E -140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134 Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; -73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F -115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109 Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; -73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K -90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-S7; Q73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q -73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which is at least 80%, 85%, 90% identical to the polynucleotide sequence encoding the Neutrokine-α and/or Neutrokine-αSV polypeptide described above %, 92%, 95%, 96%, 97%, 98% or 99% identity, the present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides comprise or consist of at least 80%, 85%, 90%, 92%, 95%, 96%, Polypeptides consisting of amino acid sequences with 97%, 98% or 99% identity, and polynucleotides encoding these polypeptides are also included in the present invention.

The present invention also provides a polypeptide having one or more amino acids deleted from the amino-terminal and carboxy-terminal ends of the putative ectodomain of Neutrokine-α, which can be described as having n ² -m ² residues of SEQ ID NO: 2, wherein n ² and m ² is an integer as described above.

In another embodiment, a nucleotide sequence encodes a polypeptide consisting of a part of the ectodomain of the Neutrokine-α amino acid sequence, the Neutrokine-α amino acid sequence is encoded by the cDNA plasmid No. 97768 in ATCC, and the part of the ectodomain is not Including the 1-206 amino acid at the amino terminal of the ectodomain of the Neutrokine-α amino acid sequence, or the 1-206 amino acid at the carboxy terminal, or any combination of the above amino terminal and carboxyl terminal deletions.

As noted above, even if one or more amino acids are deleted from the N-terminus of a polypeptide resulting in the loss of one or more functional activities (ie, biological activities) of the polypeptide, other functions or biological activities may still be retained. Thus, the ability of the shortened Neutrokine-alpha mutein to induce and/or bind antibodies that recognize the full-length or mature form of the polypeptide or its ectodomain remains unchanged when a small number of residues of the full-length or mature polypeptide or its ectodomain are removed from the N-terminus. Can be reserved. Whether a particular polypeptide lacking the N-terminal residues of the complete polypeptide retains such immunological activity can be determined by methods described herein and known in the art. It is not impossible that a Neutrokine-alpha mutein missing a large number of N-terminal amino acid residues still retains some functional (eg biological or immunological) activity. In fact, peptides consisting of as few as 6 Neutrokine-alpha amino acid residues can often elicit an immune response as well.

Therefore, the present invention provides a polypeptide and a polynucleotide encoding such a polypeptide, the polypeptide having a amino acid missing from the amino terminal of the deduced full-length amino acid sequence of Neutrokine-α shown in SEQ ID NO: 2 up to the 280th glycine residue one or more residues. In particular, the present invention provides a polypeptide comprising an amino acid sequence consisting of n ³ -285 residues of the sequence shown in SEQ ID NO: 2, wherein n ³ is one of the 1-280 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 integer.

More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: D-2 to L-285 of SEQ ID NO: 2; D-3 to L- 285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; 16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; L-285; K23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E- 35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L- 54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; L-283; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; 285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; 79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; 285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L285; 98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; 285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; 123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; L-285; R130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; 142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; 285; T-155 to L-285; P-156 to L-285; T-157 to L-283; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; 167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L- 285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; 192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L- 285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V- 217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; 285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; 242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L- 285; E-255 to L-285; G-256 to L-285; D-257 to L-295; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; 1-263 to L-285; P-264 to L-285; R-265 to L-285; 267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; 285; and G-280 to L-285. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which is at least 80%, 85%, 90% identical to the polynucleotide sequence encoding the above-mentioned Neutrokine-α and/or Neutrokine-αSV polypeptide %, 92%, 95%, 96%, 97%, 98% or 99% identity, the present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, and polynucleotides encoding these polypeptides are also encompassed by the present invention.

As described above, even if one or more amino acids are deleted from the C-terminus of a protein, resulting in the loss of one or more functional activities (eg, biological activities) of the protein, other functional activities may still be retained. Thus, the ability of the shortened Neutrokine-alpha muteins to induce and/or bind antibodies recognizing the intact or mature polypeptide or ectodomain may be retained when a small number of residues of the intact or mature polypeptide or ectodomain are removed from the C-terminus. Whether a particular polypeptide retains such immunological activity by deletion of the C-terminus of the intact polypeptide can be determined by the methods described herein and other methods known in the art. It is not impossible that a Neutrokine-α mutein missing a large number of C-terminal amino acid residues still retains some functional (eg biological or immunological) activity. In fact, peptides consisting of as few as 6 Neutrokine-alpha amino acid residues can often elicit an immune response as well.

Therefore, the present invention provides a polypeptide having one or more residues deleted from the carboxy-terminal of the Neutrokine-α amino acid sequence shown in SEQ ID NO: 2 up to glutamic acid at position 6, and a polynuclear polynucleotide encoding this polypeptide is also provided. glycosides. In particular, the present invention provides a polypeptide comprising an amino acid sequence consisting of 1- ^m3 residues of SEQ ID NO: 2, wherein ^m3 is an integer position in amino acid residue positions 6-284 of the amino acid sequence shown in SEQ ID NO: 2.

More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: M-1 to L-284 of SEQ ID NO: 2; M-1 to K-283 M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; -1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P -264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258 M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; -1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T -239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233 ;M-1 to C-232;M-1 to R-231;M-1 to F-230;M-1 to L-229;M-1 to T-228;M-1 to V-227;M -1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-118; M-1 to V-117; M-1 to K-216; M-1 to K-115; M-1 to R -114; M-1 to Q-113; M-1 to I-112; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208 ;M-1 to A-207;M-1 to Y-206;M-1 to T-205;M-1 to K-204;M-1 to D-203;M-1 to T-202;M -1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to 1-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E -189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183 M-1 to E-132; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; -1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to T -164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158 M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; -1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E -139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133 M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; -1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114 ;M-1 to K-113;M-1 to L-112;M-1 to G-111;M-1 to A-110;M-1 to T-109;M-1 to V-108;M -1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A -95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89 M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-35; M-1 to Q-84; M-1 to L-83; -1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A -70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64 M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M -1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G -45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39 M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; -1 to P-32; M-1 to L-31; M-1 to 1-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E -20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14 M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M -1 to R-7; and M-1 to E-6. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which is at least 80%, 85%, 90% identical to the polynucleotide sequence encoding the Neutrokine-α and/or Neutrokine-αSV polypeptide described above %, 92%, 95%, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, the polypeptides comprising at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, and polynucleotides encoding such polypeptides are also encompassed by the present invention.

The present invention also provides a polypeptide having one or more amino acids deleted from the amino and carboxy termini of a Neutrokine-alpha polypeptide, which can be described as having residues n ³ -m ³ of SEQ ID NO: 2, wherein n ³ and m ³ is an integer as described above.

In addition, since the putative ectodomain of the Neutrokine-αSV polypeptide of the present invention can stimulate functional activity (such as biological activity) by itself, the N and C-terminal amino acid deletions of the putative ectodomain of the polypeptide at Gln73-Leu266 of SEQ ID NO: 19 can still be retained Some functional activities, such as ligand binding, stimulate lymphocyte (eg, B cell) proliferation, differentiation and/or activation, regulate cell replication, modulate target cell activity and/or immune activity. However, even if deletion of one or more amino acids from the N-terminus of the putative ectodomain of a Neutrokine-αSV polypeptide results in loss of one or more functional activities, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind antibodies that recognize the full or mature polypeptide or its ectodomain may be retained when a small number of residues of the mature or full polypeptide or ectodomain are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residues of the complete polypeptide retains such immunological activity can be determined by routine methods described herein and other methods known in the art.

Therefore, the present invention also provides a polypeptide and a polynucleotide encoding the polypeptide, the polypeptide has one or more residues deleted from the amino terminal of the Neutrokine-αSV amino acid sequence up to the 261st glycine residue. In particular, the present invention provides a polypeptide comprising the n ⁴ -266 amino acid sequence of SEQ ID NO: 19, wherein n ⁴ is an integer position in the amino acid residue positions 73-261 of the amino acid sequence of SEQ ID NO: 19, and position 261 is Neutrokine - the first residue at the N-terminus of the putative ectodomain of the alphaSV (shown in SEQ ID NO: 19) polypeptide.

More particularly, in certain embodiments, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: Q-73 to L- of SEQ ID NO: 19 266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L79 to L-266; R- 80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266, G-85 to L-266; L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; 266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; 105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; 266; P-118 to L-266; A119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N- 124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R130 to L- 266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; 143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L- 266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; 162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L- 266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; 187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L- 266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-203 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; 212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L- 266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; 237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-233 to L-266; D-254 to L-266; G-255 to L-266; 256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to L-266. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence having at least 80%, 85%, 90%, 92%, 95% identity with the polynucleotide sequence encoding the Neutrokine-αSV polypeptide described above. %, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, the polypeptides comprising at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, and polynucleotides encoding such polypeptides are also encompassed by the present invention.

Similarly, the deletion of the C-terminal amino acids of the putative ectodomain of Neutrokine-αSV up to the 79th leucine of SEQ ID NO: 19 may retain some functional activities, such as ligand binding, stimulation of lymphocyte (such as B cell) proliferation, differentiation and/or activation, modulation of cell replication, modulation of target cell viability and/or immunogenicity. Polypeptides having further C-terminal deletions of SEQ ID NO: 19 including deletions of Leu79 are not expected to retain biological activity.

However, even if the deletion of one or more amino acids from the C-terminus of a polypeptide results in the loss of one or more functional activities (eg, biological activities) of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind antibodies that recognize the full polypeptide, the mature polypeptide or its ectodomain may be retained when a small number of residues of the mature, full polypeptide or ectodomain are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residues of a putative ectodomain retains such immunological activity can be determined by routine methods described herein and other methods known in the art.

Therefore, the present invention further provides a polypeptide and a polynucleotide encoding the polypeptide, the polypeptide has an amino acid sequence deleted from the carboxyl-terminus of the putative ectodomain of Neutrokine-αSV shown in SEQ ID NO: 19 up to Leucine 79 of one or more residues. In particular, the present invention provides a polypeptide having an amino acid sequence consisting of 73-m ⁴ residues of the amino acid sequence shown in SEQ ID NO: 19, wherein m ⁴ is the 79-266 amino acid residue of the amino acid sequence shown in SEQ ID NO: 19 An integer bit in the base.

More particularly, in certain embodiments, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: Q-73 to K-264 of SEQ ID NO: 19; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; 73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; 240; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q73 to E-239; 73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C- 226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; 73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; 201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; 73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; 176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q- 73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L- 151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; 73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; 126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; 73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G- 101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; 73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79; and Q-73 to S-78. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which shares at least 80%, 85%, 90%, 92%, 95% with the polynucleotide sequence encoding the above-mentioned Neutrokine-αSV polypeptide %, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and such polypeptides contain at least 80%, 85%, 90%, 92%, 95%, 96%, 97% of the above amino acid sequences , 98% or 99% identical amino acid sequences, and polynucleotides encoding such polypeptides are also included in the present invention.

The present invention also provides a polypeptide having one or more amino acids deleted from the putative ectodomain amino-terminal and carboxyl-terminal of Neutrokine-αSV, which can be described as having residues n ⁴ -m ⁴ of SEQ ID NO: 19, wherein n ⁴ and m ⁴ are integers as previously described.

In another embodiment, a nucleotide sequence encodes a polypeptide consisting of a portion of the extracellular domain of the Neutrokine-αSV amino acid sequence encoded by the deposited cDNA clone of ATCC Accession No. 203518, the portion of which is not Including amino acids 1-260 at the amino terminal of the extracellular domain, or amino acids 1-187 at the carboxyl terminal of the extracellular domain, or any combination of the above amino-terminal and amino-terminal deletions of the extracellular domain.

As noted above, even if one or more amino acids are deleted from the N-terminus of a polypeptide resulting in loss of one or more functional activities (eg, biological activities) of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened Neutrokine-αSV mutein to induce and/or bind antibodies recognizing the full-length or mature polypeptide or its ectodomain is retained when a small number of residues of the full-length or mature polypeptide or its ectodomain are removed from the N-terminus . Whether a particular polypeptide lacking the N-terminal residues of the complete polypeptide retains such immunological activity can be determined by routine methods described herein and other methods known in the art. It is not impossible that a Neutrokine-αSV mutein with substantial deletion of N-terminal amino acid residues would still retain functional (eg, immunogenic) activity. In fact, peptides consisting of as few as 6 amino acid residues of Neutrokine-αSV also commonly elicit an immune response.

Therefore, the present invention provides a polypeptide and a polynucleotide encoding such a polypeptide, which has one or more amino acids missing from the amino terminal of the deduced full-length amino acid sequence of Neutrokine-αSV shown in SEQ ID NO: 19 up to glycine at position 261. multiple residues. In particular, the present invention provides an amino acid sequence comprising residues n ^5-266 of the sequence shown in SEQ ID NO: 19, wherein m ⁵ is the amino acid sequence of residues 1-261 of the amino acid sequence shown in SEQ ID NO: 19 - Integer bits.

More particularly, the present invention provides a polynucleotide encoding a polypeptide selected from the amino acid sequence of the following group: D-2 to L-266 of SEQ ID NO: 19; D-3 to L-266; S- 4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L- 266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266, C-27 to L-266; V-28 to L-266; 29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L- 266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L- 54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L- 266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; 79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; L-266; H-36 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; 266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; 104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L- 266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; 129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L- 266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; 154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; 266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; 179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; 266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; 204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; 1-214 to L-266; Q-215 to L-266; N-216 to L- 266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-223 to L-266; 229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L- 266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R246 to L-266; E-247 to L-266; 248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; U-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L- 266; and G-261 to L-266. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which is at least 80%, 85%, 90% identical to the polynucleotide sequence encoding the Neutrokine-α and/or Neutrokine-αSV polypeptide described above, 92%, 95%, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides have at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, polynucleotides encoding these polypeptides are also within the present invention.

As noted above, even if one or more amino acids are deleted from the C-terminus of a protein resulting in the loss of one or more functional activities of the protein (eg, biological activity), other functional activities may still be retained. Thus, the ability of the shortened Neutrokine-αSV mutein to induce and/or bind antibodies recognizing the intact or mature polypeptide or its ectodomain may be retained when a small number of residues of the intact or mature polypeptide or ectodomain are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residues of the complete polypeptide retains such immunological activity can be determined by routine methods described herein and other methods known in the art. It is not impossible that Neutrokine-αSV muteins with large deletions of C-terminal amino acid residues still retain some functional (eg, immunogenicity) activity. In fact, peptides consisting of as few as 6 amino acid residues of Neutrokine-αSV can often elicit an immune response as well.

Therefore, the present invention provides a polypeptide and a polynucleotide encoding the polypeptide. The polypeptide has one or more amino acids deleted from the carboxy-terminal of the Neutrokine-αSV amino acid sequence shown in SEQ ID NO: 19 to the glutamic acid at position 6. multiple residues. In particular, the present invention provides a polypeptide comprising the 1- ^m5 residue amino acid sequence of SEQ ID NO: 19, wherein m ⁵ is an integer position in the 6-265 amino acid residue positions of the amino acid sequence shown in SEQ ID NO: 19.

More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: M-1 to G-265 of SEQ ID NO: 19; M-1 to G-264 ;L-263;M-1 to A-262;M-1 to G-261;M-1 to F-260;M-1 to F-259;M-1 to T-258;M-1 to V -257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251 ;M-1 to Q-250;M-1 to A-249;M-1 to N-248;M-1 to E-247;M-1 to R-246;M-1 to R-245;M -1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235, 5M-1 to L-234; M-1 to K-233; M-1 to A -232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226 ;M-1 to S-225;M-1 to N-224;M-1 to N-223;M-1 to P-222;M-1 to L-221;M-1 to T-220;M -1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-115; M-1 to I-214; M-1 to C-213; M-1 to R-112; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L -207; M-10 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201 ;M-1 to V-200;M-1 to H-199;M-1 to V-198;M-1 to K-197;M-1 to K-196;M-1 to R-195;M -1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y -182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176 ;M-1 to F-175;M-1 to F-174;M-1 to Y-173;M-1 to G-172;M-1 to T-171;M-1 to E-170;M -1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to 1-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S -157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151 ;M-1 to L-150;M-1 to W-149;M-1 to P-148;M-1 to V-147;M-1 to F-146;M-1 to T-145;M -1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K -132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126 ;M-1 to S-125;M-1 to N-124;M-1 to G-123;M-1 to E-122;M-1 to G-121;M-11 to P-120;M -1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-110; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A -107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101 M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M -1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E -82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76 M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M -1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L -57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51 M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M -1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P -32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26 M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M -1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R -7; and M-1 to E-6. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, polynucleotides encoding these polypeptides are also within the present invention.

The invention also provides polypeptides having one or more amino acids deleted from the amino and carboxy termini of the Neutrokine-αSV polypeptide, which can be described as having n ⁵ -m ⁵ residues of SEQ ID NO: 19, wherein n ⁵ and m ⁵ is the above integer.

In another embodiment, the present invention provides a polypeptide comprising the amino acid sequence of residues 134- ^m6 of SEQ ID NO:2, wherein ^m6 is amino acid residues 140-285 of the sequence shown in SEQ ID NO:2 An integer in . For example, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: A-134 to Leu-285 of SEQ ID NO: 2; A-134 to L-284; A- 134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; 271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; 134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; 246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; 134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G- 221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; 134 to M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; 196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; 134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134 to K-173; A-134 to F-172; 171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; 134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; 146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A-134 to V-142; A-134 to T-141; and A-134 to E-140 . The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence having at least 80%, 85%, 90% identity with the polynucleotide sequence encoding the Neutrokine-α and/or Neutrokine-αSV polypeptide described above , 92%, 95%, 96%, 97%, 98% or 99% identity. The present invention also covers the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, and these polypeptides have at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% identity, polynucleotides encoding these polypeptides are also within the present invention.

Other preferred polypeptide fragments of the present invention comprise or consist of amino acid sequences selected from the following group: M-1 to C-15 of SEQ ID NO: 2; D-2 to L-16; D-3 to K-17; S -4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I -30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36 ;K-23 to P-37;L-24 to S-38;K-25 to V-39;E-26 to R-40;C-27 to S-41;V-28 to S-42;S -29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L48; E-35 to A -49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55 ;S-42 to L-56;K-43 to L-57;D-44 to S-58;G-45 to C-59;K-46 to C-60;L-47 to L-61;L -48 to T-62; A-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G -74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80 ;Y-67 to A-81;Q-68 to E-82;V-69 to L-83;A-70 to Q-84;A-71 to G-85;L-72 to H-86;Q -73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K -99; H-86 to A-100; H-87 to G-101; A-83 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105 ; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; -98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-I17; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-103 to E-122; T-109 to G-123; A-110 to N -124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-110 to S-129; E-116 to R-130 ; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; -123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-123 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L -149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155 ; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; -148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R -174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180 ;P-167 to K-181;W-168 to E-182;L-169 to N-183;L-170 to K-184;S-171 to iI-185;F-172 to L-186;K -173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V -199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205 ; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; -198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L -224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230 ; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; -223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G -249; M-236 to 1-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255 ; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; -248 to A-262; G249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A -268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274 ;L-261 to D-275;A-262 to V-276;I-263 to T-277;P-264 to F-278;R-265 to F-279;E-266 to G-280;N -267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284; and S-271 to L-285. Preferably, these polypeptide fragments have one or more functional activities (such as biological activity, antigenicity and immunogenicity) of the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention, and can be used, for example, to generate or screen antibodies, as follows mentioned. The invention also relates to amino acid sequences comprising or consisting of at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the aforementioned amino acid sequences. The present invention also covers the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other preferred polypeptide fragments of the present invention comprise or consist of amino acid sequences selected from the following group: M-1 to C-15 of SEQ ID NO: 19; D-2 to L-16; D-3 to K-17; S -4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I -30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36 ;K-23 to P-37;L-24 to S-38;K-25 to V-39;E-26 to R-40;C-27 to S-41;V-28 to S-42;S -29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A -55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-43 to C-59; K-46 to C-60; L-47 to L-61 ;L-48 to T-62;A-49 to V-63;A-50 to V-64;T-51 to S-65;L-52 to F-66;L-53 to Y-67;L -54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-73; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R -80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86 ; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; -79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A -105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111 ; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; -104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R -130; P-I17 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136 ; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; -129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to F-146; R-133 to V-147; to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R -155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161 ; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; -154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; I-166 to V -180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186 ; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; -179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L -205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211 ; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-213; G-202 to N-216; -204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G -230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236 ; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; -229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-24S; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G -250; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261 ; N-248 to A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265; and S-252 to L-266. Preferably, these polypeptide fragments have one or more functional activities (such as biological activity, antigenicity and immunogenicity) of the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention, and can be used, for example, to generate or screen antibodies, as follows mentioned. The invention also relates to polypeptides comprising or consisting of amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences. The present invention also covers the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other preferred polypeptide fragments of the present invention comprise or consist of amino acid sequences selected from the following group: M-1 to F-15 of SEQ ID NO: 38; D-2 to C-16; E-3 to S-17; S -4 to E-18; A-5 to K-19; K-6 to G-20; T7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K -24; P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30 ;S-17 to T-31;E-18 to P-32;K-19 to Q-33;G-20 to K-34;E-21 to E-35;D-22 to E-36;M -23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L -49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to L-55 ;I-42 to A-56;C-43 to L-57;R-44 to L-58;D-45 to S-59;G-46 to S-60;R-47 to S-61;L -48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q -74; S-61 to A-73; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80 ;L-67 to R-81;Y-68 to M-82;Q-69 to E-83;L-70 to L-84;A-71 to Q-85;A-72 to S-86;L -73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P -99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105 ; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-93 to T-109; A-96 to P-110; -98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N -124; A-111 to 125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N -117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P -143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149 D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-105; A -142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to I-165; R-152 to I-166; H-153 to I-167; S-154 to Q -168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-174 ;M-161 to 175;N-162 to D-176;L-163 to S-177;R-164 to D-178;N-165 to T-179;I-166 to P-180;I-167 to A-181; Q-163 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N -187; I-174 to K-188; A-175 to I-189; D-176 to V-191; S-177 to V-191; D-178 to R-192; T-179 to Q-193 ; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; -186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-203; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M -212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218 ;Y-205 to K-219;T-206 to K-220;D-207 to V-221;P-208 to H-222;I-209 to V-223;F-210 to F-224;A -211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to 1 -237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243 ; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; -236 to Y-250; I-237 to S-250; Q-238 to A-252; N-239 to G-253; M-240 to I-254; to R-256; T-243 to L-257; L-244 to E-253; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E -262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-234 to P-268 ;A-255 to R-269;R-256 to E-270;L-257 to N-271;E-258 to A-272;E-259 to Q-273;G-260 to I-274;D -261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-272 to L-286; Q-273 to K -287; I-274 to L-288; and S-275 to L-289. Preferably, these polypeptide fragments have one or more activities of the Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention, and can be used, for example, to generate or screen antibodies, as described below. The invention also relates to polypeptides comprising or consisting of amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences. The present invention also covers the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Those skilled in the art recognize that the amino acid sequence of some Neutrokine-alpha and Neutrokine-alpha SV polypeptides can be altered without appreciably affecting the structure or function of the polypeptide. If such sequence differences are desired, it should be remembered that there are critical regions that determine the activity of the polypeptide.

Accordingly, the present invention also provides variants of Neutrokine-alpha polypeptides that exhibit functional activity (eg, biological activity) of Neutrokine-alpha polypeptides or regions thereof comprising Neutrokine-alpha polypeptides, such as polypeptide fragments described herein. The invention also includes variants of Neutrokine-αSV polypeptides that exhibit the functional activity (eg, biological activity) of Neutrokine-αSV polypeptides, or regions thereof that include Neutrokine-αSV polypeptides, such as polypeptide fragments described herein. Such mutants include insertions, deletions, transitions, duplications and pattern substitutions selected according to general rules known in the art and thus have less effect on activity. For example, guidance on how to produce phenotypically silent amino acid substitutions is found in Bowie, J.U. et al., "Interpretation of Information in Protein Sequences Resistant to Amino Acid Substitutions", Science 247:1306-1310 (1990), where the authors state that there are two main approaches to Amino acid sequence tolerance to variation. The first approach relies on the process of evolution, in which mutations are accepted or repelled by natural selection. The second approach is to use genetic engineering to make amino acid changes at specific locations in the cloned gene and perform selection or screening to identify sequences that remain functional.

As stated by the authors, these studies have demonstrated that proteins are surprisingly resistant to amino acid substitutions. The authors also indicate that amino acid changes are permissible at some positions in the protein. For example, most cryptic amino acid residues require nonpolar side chains, whereas surface side chains are often conserved. Other such phenotypically silent substitutions are described in Bowie, J.U. et al., supra, and in references indicated herein. Obvious conservative substitutions are those between the aliphatic amino acids Ala, Val, Leu and Ile; between the hydroxyl residues Ser and Thr, between the acidic residues Asp and Glu, between the amide residues Asn and Gln The substitution between the basic residues Lys and Arg, and the substitution between the aromatic residues Phe, Tyr.

Accordingly, the polypeptide shown in Figures 1A and 1B (SEQ ID NO: 2) or a fragment, derivative or analogue of the polypeptide encoded by the deposited cDNA plasmid may be one of the following: (i) wherein one or more amino acid residues are represented by Conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code; (ii) wherein one or more amino acid residues include a substitution or (iii) wherein the ectodomain of the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); or (iv) wherein other amino acids are fused to the ectodomain of the polypeptide, such as an IgG FC fusion region Peptide or leader or secretory sequence or sequence for purification of polypeptide ectodomain or preprotein sequence. Such fragments, derivatives and analogs are considered to be within the scope of the present invention by those skilled in the art given the teachings herein.

In addition, a fragment, derivative or analogue of the polypeptide shown in Figures 5A and 5B (SEQ ID NO: 19) or encoded by the deposited cDNA plasmid may be one in which (i) one or more amino acid residues Substitution with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) wherein one or more amino acid residues include A substituent, or (iii) wherein the ectodomain of the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g. polyethylene glycol), or (iv) wherein other amino acids are fused to the ectodomain of the polypeptide, such as other Soluble bioactive fragments of TNF ligand family members (such as CD40 ligands), IgGF _C- fusion region peptides or leader or secretory sequences, or sequences used to purify the extracellular domain of polypeptides or preprotein sequences. Such fragments, derivatives and analogs are considered to be within the scope of the present invention by those skilled in the art given the teachings herein.

Therefore, the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, which are natural mutations or artificial manipulations. As noted above, changes are preferably minor, such as conservative amino acid substitutions that do not substantially affect protein folding or activity (see Table 2).

Table 2 Conservative amino acid substitutions

Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polarity Glutamine Asparagine Alkaline Arginine Lysine Histidine acidic Aspartic Acid Glutamic Acid Small molecule Alanine Serine Threonine Methionine Glycine

In one embodiment of the present invention, the polypeptide comprises or consists of the amino acid sequence of Neutrokine-α and/or Neutrokine-αSV polypeptide, the amino acid sequence of the Neutrokine-α and/or Neutrokine-αSV polypeptide contains at least 1 but not more than 50 , preferably no more than 40, more preferably no more than 30, and most preferably no more than 20 conservative amino acid substitutions. Of course, it is most preferred to contain at least 1 but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.

For example, site-directed mutagenesis at the amino acid level of Neutrokine-alpha can be performed by replacing specific amino acids with conservative substituents. Preferred conservative substitution mutations in the Neutrokine-α amino acid sequence shown in SEQ ID NO: 2 include: replace M1 with A, G, I, L, S, T or V; use A, G, I, L, T, or V Replace M1; replace D2 with E; replace D3 with E; replace S4 with A, G, I, L, T, M or V; replace T5 with A, G, I, L, S, M or V; replace D E6; replace R7 with H, or K; replace E8 with D; replace Q9 with N; replace S10 with A, G, I, L, T, or V; replace R11 with H, or K; use A, G, I , S, T, M or V for L12; A, G, I, L, S, M or V for T13; A, G, I, L, T, M or V for S14; A, G, I, S, T, M or V to replace L16; use H, or R to replace K17; use H, or R to replace K18; use H, or K to replace R19; use D to replace E20; use D to replace E21; use A, G , I, L, S, T, replace M22; replace K23 with H, or R; replace L24 with A, G, I, S, T, M or V; replace K25 with H, or R; replace E26 with D; Replace V28 with A, G, I, L, S, T or M; replace S29 with A, G, I, L, T, M or V; replace I30 with A, G, L, S, T, M or V ; Replace L31 with A, G, I, S, T, M or V; Replace R33 with H, or K; ; Replace K34 with H, or R; Replace E35 with D; Use A, G, I, L, T , M or V to replace S36; use A, G, I, L, T, M or V to replace S38; use A, G, I, L, S, T or M to replace V39; use H, or K to replace R40; A, G, I, L, T, M or V to replace S41; use A, G, I, L, T, M or V to replace S42; use H, or R to replace K43; use E to replace D44; use A, I , L, S, T, M or V for G45; H, or R for K46; A, G, I, S, T, M or V for L47; A, G, I, S, T, M or V to replace L48; use G, I, L, S, T, M or V to replace A49; use G, I, L, S, T, M or V to replace A50; use A, G, I, L, S, Replace T51 with M or V; replace L52 with A, G, I, S, T, M, or V; replace L53 with A, G, I, S, T, M, or V; replace A, G, I, S, Replace L54 with T, M or V; replace A55 with G, I, L S, T, M or V; replace L56 with A, G, I, S, T, M or V; replace A, G, I, S, Replace L57 with T, M or V; replace S58 with A, G, I, L, T, or V; replace L61 with A, G, I, L, S, T, M or V; replace A, G, I, L, S, M or V for T62; A, G, I, L, S, T, or M for V63; A, G, I, L, S, T, or M for V64; A, G , I, L, T, M, or V to replace S65; use W, or Y to replace F66; use F, or W to replace Y67; use N to replace Q68; use A, G, I, L, S, T, or M Replace V69; replace A70 with G, I, L S, T, M or V; replace A71 with G, I, L S, T, M or V; replace A, G, I, S, T, M or V L72; replace Q73 with N; replace G74 with A, I, L, S, T, M or V; replace D75 with E; replace L76 with A, G, I, S, T, M or V; use G, I , L, S, T, M or V to replace A77; use A, G, I, L, T, M or V to replace S78; use A, G, I, S, T, M or V to replace L79; use H, Or K to replace R80; use G, I, L, S, T, M or V to replace A81; use D to replace E82; use A, G, I, S, T, M or V to replace L83; use N to replace Q84; A, I, L, S, T, M or V for G85; K or R for H86; K or R for H87; G, I, L S, T, M or V for A88; D for E89 ; Replace K90 with H or R; Replace L91 with A, G, I, S, T, M or V; Replace A93 with G, I, L S, T, M or V; Replace A, I, L, S, T, M or V for G94; G, I, L S, T, M or V for A95; A, I, L, S, T, M or V for G96; G, I, L S, T , M or V to replace A97; use H or R to replace K99; use G, I, L S, T, M or V to replace A100; use A, I, L, S, T, M or V to replace G101; use A, G, I, S, T, M or V for L102; D for E103; D for E104; G, I, L S, T, M or V for A105; G, I, L S, T, Replace A107 with M or V; replace V108 with A, G, I, L, S, T or M; replace T109 with A, G, I, L, S, M or V; replace G, I, L S, T, Replace A110 with M or V; replace G111 with A, I, L, S, T, M or V; replace L112 with A, G, I, S, T, M or V; replace K113 with H or R; G, L, S, T, M or V for I114; W or Y for F115; D for E116; G, I, L S, T, M or V for A119; A, I, L, S , T, M or V for G121; D for E122; A, I, L, S, T, M or V for G123; Q for N124; A, G, I, L, T, M or V Replace S125; replace S126 with A, G, I, L, T, M or V; replace Q127 with N; replace N128 with Q; replace S129 with A, G, I, L, T, M or V; use H, or K to replace R130; use Q to replace N131; use H or R to replace K132; use H or K to replace R133; use G, I, L S, T, M or V to replace A134; use A, G, I, L, Replace V135 with S, T or M; replace Q136 with N; replace G137 with A, I, L, S, T, M or V; replace E139 with D; replace E140 with D; use A, G, I, L, S , M or V for T141; A, G, I, L, S, T or M for V142; A, G, I, L, S, M or V for T143; N for Q144; E for D145 ; Substitute A, G, I, S, T, M or V for L147; Substitute N for Q148; Substitute A, G, I, S, T, M or V for L149; Substitute A, G, L, S, T , M or V to replace I150; use G, I, L S, T, M or V to replace A151; use E to replace D152; use A, G, I, L, T, M or V to replace S153; use D to replace E154; Replace T155 with A, G, I, L, S, M or V; replace T157 with A, G, I, L, S, M or V; replace I158 with A, G, L, S, T, M or V ; replace Q159 with N; replace K160 with H or R; replace G161 with A, I, L, S, T, M or V; replace S162 with A, G, I, L, T, M or V; use F or Replace Y163 with W; replace T164 with A, G, I, L, S, M or V; replace F165 with W or Y; replace V166 with A, G, I, L, S, T or M; replace F or Y W168; replace L169 with A, G, I, S, T, M or V; replace L170 with A, G, I, S, T, M or V; replace A, G, I, L, T, M or V Replace S171; replace F172 with W or Y; replace K173 with H or R; replace R174 with H or K; replace G175 with A, I, L, S, T, M or V; use A, G, I, L, Replace S176 with T, M or V; replace A177 with G, I, L S, T, M or V; replace L178 with A, G, I, S, T, M or V; replace E179 with D; replace E180 with D ; replace K181 with H or R; replace E182 with D; replace N183 with Q; replace K184 with H or R; replace I185 with A, G, L, S, T, M or V; use A, G, I, S , T, M or V for L186; A, G, I, L, S, T or M for V187; H or R for K188; D for E189; A, G, I, L, S, M Or V to replace T190; use A, I, L, S, T, M or V to replace G191; use F or W to replace Y192; use W or Y to replace F193; use W or Y to replace F194; use A, G, L, S, T, M or V for I195; F or W for Y196; A, I, L, S, T, M or V for G197; N for Q198; A, G, I, L, S, Replace V199 with T or M; replace L200 with A, G, I, S, T, M or V; replace Y201 with F or W; replace T202 with A, G, I, L, S, M or V; replace with E D203; replace K204 with H or R; replace T205 with A, G, I, L, S, M or V; replace Y206 with F or W; replace A207 with G, I, L S, T, M or V; A, G, I, L, S, T or V for M208; A, I, L, S, T, M or V for G209; K or R for H210; A, G, I, S, T , replace L211 with M or V; replace I212 with A, G, L, S, T, M or V; replace Q213 with N; replace R214 with H or K; replace K215 with H or R; replace K216 with H or R; Replace V217 with A, G, I, L, S, T or M; replace H218 with K or R; replace V219 with A, G, I, L, S, T or M; replace F220 with W or y; , I, L, S, T, M or V for G221; E for D222; D for E223; A, G, I, S, T, M or V for L224; A, G, I, L , T, M or V for S225; A, G, I, S, T, M or V for L226; A, G, I, L, S, T or M for V227; A, G, I, Replace T228 with L, S, M or V; replace L229 with A, G, I, S, T, M or V; replace F230 with W or Y; replace R231 with H or K; use A, G, L, S, Replace I233 with T, M or V; replace Q234 with N; replace N235 with Q; replace M236 with A, G, I, L, S, T or V; replace E238 with D; use A, G, I, L, S , M or V for T239; A, G, I, S, T, M or V for L240; Q for N242; Q for N243; A, G, I, L, T, M or V for S244 ; Replace Y246 with F or W; Replace S247 with A, G, I, L, T, M or V; Replace A248 with G, I, L S, T, M or V; Replace A, I, L, S, Replace G249 with T, M or V; replace I250 with A, G, L, S, T, M or V; replace A251 with G, I, L S, T, M or V; replace K252 with H or R; Replace L253 with A, G, I, S, T, M or V; replace E254 with D; replace E255 with D; replace G256 with A, I, L, S, T, M or V; replace D257 with E; Replace E258; replace L259 with A, G, I, S, T, M or V; replace Q260 with N; replace L261 with A, G, I, S, T, M or V; use G, I, L S, T, M or V for A262; A, G, L, S, T, M or V for I263; H or K for R265; D for E266; Q for N267; G, I, L, S , T, M or V for A268; N for Q269; A, G, L, S, T, M or V for I270; A, G, I, L, T, M or V for S271; , G, I, S, T, M or V for L272; E for D273; A, I, L, S, T, M or V for G274; E for D275; A, G, I, L , S, T or M for V276; A, G, I, L, S, M or V for T277; W or Y for F278; W or Y for F279; A, I, L, S, T , M or V to replace G280; use G, I, L S, T, M or V to replace A281; use A, G, I, S, T, M or V to replace L282; use H or R to replace K283; use A, G, I, S, T, M or V in place of L284; and/or A, G, I, S, T, M or V in place of L285. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (eg, enhanced or decreased stability and/or solubility). Preferably, the protein obtained in the present invention has increased and/or decreased functional activity of Neutrokine-α and/or Neutrokine-αSV. More preferably, the Neutrokine-α and/or Neutrokine-αSV protein obtained in the present invention has more than one increased and/or decreased functional activity and/or physical properties of Neutrokine-α and/or Neutrokine-αSV.

In another embodiment, site-directed mutagenesis at the amino acid level of Neutrokine-αSV can be performed by replacing specific amino acids with conservative substituents. The preferred conservative substitution mutations of the Neutrokine-αSV amino acid sequence shown in SEQ ID NO: 19 include: replacing M1 with A, G, I, L, S, T or V; replacing D2 with E; replacing D3 with E; replacing D3 with A, G , I, L, T, M or V for S4; A, G, I, L, S, M or V for T5; D for E6; H or K for R7; D for E8; Q9; replace S10 with A, G, I, L, T, M or V; replace R11 with H or K; replace L12 with A, G, I, S, T, M or V; use A, G, I, Replace T13 with L, S, M or V; replace S14 with A, G, I, L, T, M or V; replace L16 with A, G, I, S, T, M or V; replace K17 with H or R ; replace K18 with H or R; replace R19 with H or K; replace E20 with D; replace E21 with D; replace M22 with A, G, I, L, S, T or V; replace K23 with H or R; A, G, I, S, T, M or V for L24; H or R for K25; D for E26; A, G, I, L, S, T or M for V28; A, G, I, L, T, M or V for S29; A, G, L, S, T, M or V for I30; A, G, I, S, T, M or V for L31; H or K Replace R33; replace K34 with H or R; replace E35 with D; replace S36 with A, G, I, L, T, M or V; replace S38 with A, G, I, L, T, M or V; A, G, I, L, S, T or M for V39; H or K for R40; A, G, I, L, T, M or V for S41; A, G, I, L, T , M or V for S42; H or R for K43; E for D44; A, I, L, S, T, M or V for G45; H or R for K46; A, G, I, S, T, M or V for L47; A, G, I, S, T, M or V for L48; G, I, L, S, T, M or V for A49; G, I, L , S, T, M or V to replace A50; use A, G, I, L, S, M or V to replace T51; use A, G, I, S, T, M or V to replace L52; use A, G, I, S, T, M or V for L53; A, G, I, S, T, M or V for L54; G, I, L, S, T, M or V for A55; A, G , I, S, T, M or V to replace L56; use A, G, I, S, T, M or V to replace L57; use A, G, I, L, T, M or V to replace S58; use A, G, I, S, T, M or V for L61; A, G, I, L, S, M or V for T62; A, G, I, L, S, T or M for V63; A , G, I, L, S, T or M for V64; A, G, I, L, T, M or V for S65; W or Y for F66; F or W for Y67; N for Q68 ; Replace V69 with A, G, I, L, S, T or M; Replace A70 with G, I, L, S, T, M or V; Replace with G, I, L, S, T, M or V A71; replace L72 with A, G, I, S, T, M or V; replace Q73 with N; replace G74 with A, I, L, S, T, M or V; replace D75 with E; use A, G , I, S, T, M or V to replace L76; use G, I, L, S, T, M or V to replace A77; use A, G, I, L, T, M or V to replace S78; use A, G, I, S, T, M or V for L79; H or K for R80; G, I, L, S, T, M or V for A81; D for E82; A, G, I, Replace L83 with S, T, M or V; replace Q84 with N; replace G85 with A, I, L, S, T, M or V; replace H86 with K or R; replace H87 with K or R; , L, S, T, M or V for A88; D for E89; H or R for K90; A, G, I, S, T, M or V for L91; G, I, L, S , T, M or V for A93; A, I, L, S, T, M or V for G94; G, I, L, S, T, M or V for A95; A, I, L, S, T, M or V for G96; G, I, L, S, T, M or V for A97; H or R for K99; G, I, L, S, T, M or V for A100 ; replace G101 with A, I, L, S, T, M or V; replace L102 with A, G, I, S, T, M or V; replace E103 with D; replace E104 with D; use G, I, Replace A105 with L, S, T, M or V; replace A107 with G, I, L, S, T, M or V; replace V108 with A, G, I, L, S, T or M; replace A, G , I, L, S, M or V to replace T109; use G, I, L, S, T, M or V to replace A110; use A, I, L, S, T, M or V to replace G111; use A, Replace L112 with G, I, S, T, M or V; replace K113 with H or R; replace I114 with A, G, L; S, T, M or V; replace F115 with W or Y; replace E116 with D; Replace A119 with G, I, L, S, T, M or V; replace G121 with A, I, L, S, T, M or V; replace E122 with D; replace A, I, L, S, T, Replace G123 with M or V; replace N124 with Q; replace S125 with A, G, I, L, T, M or V; replace S126 with A, G, I, L, T, M or V; replace Q127 with N; Replace N128 with Q; replace S129 with A, G, I, L, T, M or V; replace R130 with H or K; replace N131 with Q; replace K132 with H or R; replace R133 with H or K; , I, L, S, T, M or V for A134; A, G, I, L, S, T or M for V135; N for Q136; A, I, L, S, T, M or Replace G137 with V; replace E139 with D; replace E140 with D; replace T141 with A, G, I, L, S, M or V; replace G142 with A, I, L, S, T, M or V; , G, I, L, T, M or V for S143; F or W for Y144; A, G, I, L, S, M or V for T145; W or Y for F146; , I, L, S, T or M for V147; F or Y for W149; A, G, I, S, T, M or V for L150; A, G, I, S, T, M or Replace L151 with V; replace S152 with A, G, I, L, T, M or V; replace F153 with W or Y; replace K154 with H or R; replace R155 with H or K; , T, M or V to replace G156; use A, G, I, L, T, M or V to replace S157; use G, I, L, S, T, M or V to replace A158; use A, G, I, S, T, M or V for L159; D for E160; D for E161; H or R for K162; D for E163; Q for N164; H or R for K165; A, G, L ; S, T, M or V for I166; A, G, I, S, T, M or V for L167; A, G, I, L, S, T or M for V168; H or R for K169; replace E170 with D; replace T171 with A, G, I, L, S, M or V; replace G172 with A, I, L, S, T, M or V; replace Y173 with F or W; or Y for F174; W or Y for F175; A, G, L; S, T, M or V for I176; F or W for Y177; A, I, L, S, T, M or V Replace G178; replace Q179 with N; replace V180 with A, G, I, L, S, T or M; replace L181 with A, G, I, S, T, M or V; replace Y182 with F or W; Replace T183 with A, G, I, L, S, M or V; replace D184 with E; replace K185 with H or R; replace T186 with A, G, I, L, S, M or V; replace F or W Y187; replace A188 with G, I, L, S, T, M or V; replace M189 with A, G, I, L, S, T or V; replace A, I, L, S, T, M or V Replace G190; replace H191 with K or R; replace L192 with A, G, I, S, T, M or V; replace I193 with A, G, L; S, T, M or V; replace Q194 with N; Replace R195 with H or K; replace K196 with H or R; replace K197 with H or R; replace V198 with A, G, I, L, S, T or M; replace H199 with K or R; use A, G, I , L, S, T or M to replace V200; use W or Y to replace F201; use A, I, L, S, T, M or V to replace G202; use E to replace D203; use D to replace E204; use A, G, Replace L205 with I, S, T, M or V; Replace S206 with A, G, I, L, T, M or V; Replace L207 with A, G, I, S, T, M or V; Replace A, G, I, L, S, T or M for V208; A, G, I, L, S, M or V for T209; A, G, I, S, T, M or V for L210; W or Y to replace F211; use H or K to replace R212; use A, G, L; S, T, M or V to replace I214; use N to replace Q215; use Q to replace N216; use A, G, I, L, S, Replace M217 with T or V; replace E219 with D; replace T220 with A, G, I, L, S, M or V; replace L221 with A, G, I, S, T, M or V; replace N223 with Q; Replace N224 with Q; replace S225 with A, G, I, L, T, M or V; replace Y227 with F or W; replace S228 with A, G, I, L, T, M or V; use G, I , L, S, T, M or V for A229; A, I, L, S, T, M or V for G230; A, G, L; S, T, M or V for I231; G, Replace A232 with I, L, S, T, M or V; replace K233 with H or R; replace L234 with A, G, I, S, T, M or V; replace E235 with D; replace E236 with D; , I, L, S, T, M or V for G237; E for D238; D for E239; A, G, I, S, T, M or V for L240; N for Q241; A, G, I, S, T, M or V for L242; G, I, L, S, T, M or V for A243; A, G, L; S, T, M or V for I244; H Or K for R246; D for E247; Q for N248; G, I, L, S, T, M or V for A249; N for Q250; A, G, L; S, T, M or Replace I251 with V; replace S252 with A, G, I, L, T, M or V; replace L253 with A, G, I, S, T, M or V; replace D254 with E; use A, I, L, Replace G255 with S, T, M or V; replace D256 with E; replace V257 with A, G, I, L, S, T or M; replace T258 with A, G, I, L, S, M or V; Replace F259 with W or Y; replace F260 with W or Y; replace G261 with A, I, L, S, T, M or V; replace A262 with G, I, L, S, T, M or V; use A, G, I, S, T, M or V for L263; H or R for K264; A, G, I, S, T, M or V for L265; and/or A, G, I, S, T, M or V replaces L266. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (eg, enhanced or decreased stability and/or solubility). Preferably, the resulting protein of the invention has increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical property.

In another embodiment, site-direct mutagenesis at the level of Neutrokine-alpha amino acids can be performed by replacing specific amino acids with conservative substituents. The preferred conservative substitution mutations of the Neutrokine-α amino acid sequence shown in SEQ ID NO: 23 include: replacing R1 with H or K; replacing V2 with A, G, I, L, S, T or M; using A, G, I, Replace V3 with L, S, T or M; replace D4 with E; replace L5 with A, G, I, S, T, M or V; replace S6 with A, G, I, L, T, M or V; Replace A7 with G, I, L, S, T, M or V; replace A10 with G, I, L, S, T, M or V; replace L13 with A, G, I, S, T, M or V; Replace G15 with A, I, L, S, T, M or V; replace R17 with H or K; replace H18 with K or R; replace S19 with A, G, I, L, T, M or V; Replace Q20; replace H21 with K or R; replace D22 with E; replace D23 with E; replace N24 with Q; replace G25 with A, I, L, S, T, M or V; use A, G, I, L , S, T or V for M26; Q for N27; A, G, I, S, T, M or V for L28; H or K for R29; Q for N30; H or K for R31; Replace T32 with A, G, I, L, S, M or V; replace Y33 with F or W; replace T34 with A, G, I, L, S, M or V; replace F35 with W or Y; , G, I, L, S, T or M for V36; F or Y for W38; A, G, I, S, T, M or V for L39; A, G, I, S, T, Replace L40 with M or V; replace S41 with A, G, I, L, T, M or V; replace F42 with W or Y; replace K43 with H or R; replace R44 with H or K; , S, T, M or V for G45; Q for N46; G, I, L, S, T, M or V for A47; A, G, I, S, T, M or V for L48; ; replace E49 with D; replace E50 with D; replace K51 with H or R; replace E52 with D; replace N53 with Q; replace K54 with H or R; I55; replace V56 with A, G, I, L, S, T or M; replace V57 with A, G, I, L, S, T or M; replace R58 with H or K; replace Q59 with N; , G, I, L, S, M or V for T60; A, I, L, S, T, M or V for G61; F or W for Y62; W or Y for F63; W or Y Replace F64; replace I65 with A, G, L; S, T, M or V; replace Y66 with F or W; replace S67 with A, G, I, L, T, M or V; replace Q68 with N; A, G, I, L, S, T or M for V69; A, G, I, S, T, M or V for L70; F or W for Y71; A, G, I, L, Replace T72 with S, M or V; replace D73 with E; replace I75 with A, G, L; S, T, M or V; replace F76 with W or Y; replace G, I, L, S, T, M or Replace A77 with V; replace M78 with A, G, I, L, S, T or V; replace G79 with A, I, L, S, T, M or V; replace H80 with K or R; use A, G, I, L, S, T or M to replace V81; use A, G, L; S, T, M or V to replace I82; use N to replace Q83; use H or K to replace R84; use H or R to replace K85; use H Or R for K86; A, G, I, L, S, T or M for V87; K or R for H88; A, G, I, L, S, T or M for V89; W or Y Replace F90; replace G91 with A, I, L, S, T, M or V; replace D92 with E; replace E93 with D; replace L94 with A, G, I, S, T, M or V; G, I, L, T, M or V for S95; A, G, I, S, T, M or V for L96; A, G, I, L, S, T or M for V97; A , G, I, L, S, M or V to replace T98; use A, G, I, S, T, M or V to replace L99; use W or Y to replace F100; use H or K to replace R101; use A, G , L; S, T, M or V for I103; N for Q104; Q for N105; A, G, I, L, S, T or V for M106; H or R for K108; A, G, I, L, S, M or V for T109; A, G, I, S, T, M or V for L110; Q for N112; Q for N113; A, G, I, L, Replace S114 with T, M or V; replace Y116 with F or W; replace S117 with A, G, I, L, T, M or V; replace A118 with G, I, L, S, T, M or V; A, I, L, S, T, M or V for G119; A, G, L; S, T, M or V for I120; G, I, L, S, T, M or V for A121; Replace R122 with H or K; replace L123 with A, G, I, S, T, M or V; replace E124 with D; replace E125 with D; replace G126 with A, I, L, S, T, M or V ; Replace D127 with E; Replace E128 with D; Replace I129 with A, G, L; S, T, M or V; Replace Q130 with N; Replace L131 with A, G, I, S, T, M or V; Replace A132 with G, I, L, S, T, M or V; replace I133 with A, G, L; S, T, M or V; replace R135 with H or K; replace E136 with D; replace N137 with Q ; replace A138 with G, I, L, S, T, M or V; replace Q139 with N; replace I140 with A, G, L; S, T, M or V; replace A, G, I, L, T , M or V to replace S141; use H or K to replace R142; use Q to replace N143; use A, I, L, S, T, M or V to replace G144; use E to replace D145; use E to replace D146; use A, G , I, L, S, M or V for T147; W or Y for F148; W or Y for F149; A, I, L, S, T, M or V for G150; G, I, L , S, T, M or V for A151; A, G, I, S, T, M or V for L152; H or R for K153; A, G, I, S, T, M or V for A151 L154; and/or substitute A, G, I, S, T, M or V for L155. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (such as enhanced or decreased stability and/or solubility). Preferably, the resulting protein of the invention has increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical property.

In another embodiment, site-direct mutagenesis at the level of Neutrokine-alpha amino acids can be performed by replacing specific amino acids with conservative substituents. The preferred conservative substitution mutations of the Neutrokine-α amino acid sequence shown in SEQ ID NO: 38 include: replacing M1 with A, G, I, L, S, T or V; replacing D2 with E; replacing D3 with E; replacing D3 with A, G , I, L, T, M or V to replace S4; use G, I, L, S, T, M or V to replace A5; use H or R to replace K6; use A, G, I, L, S, M or Replace T7 with V; replace L8 with A, G, I, S, T, M or V; replace L13 with A, G, I, S, T, M or V; replace F15 with W or Y; Replace S17 with I, L, T, M or V; replace E18 with D; replace K19 with H or R; replace G20 with A, I, L, S, T, M or V; replace E21 with D; replace D22 with E ; replace M23 with A, G, I, L, S, T or V; replace K24 with H or R; replace V25 with A, G, I, L, S, T or M; use A, I, L, S , T, M or V for G26; F or W for Y27; E for D28; A, G, L; S, T, M or V for I30; A, G, I, L, S, M Or replace T31 with V; replace Q33 with N; replace K34 with H or R; replace E35 with D; replace E36 with D; replace G37 with A, I, L, S, T, M or V; use G, I, L , S, T, M or V for A38; F or Y for W39; W or Y for F40; A, I, L, S, T, M or V for G41; A, G, L; S , T, M or V for I42; H or K for R44; E for D45; A, I, L, S, T, M or V for G46; H or K for R47; A, G, Replace L48 with I, S, T, M or V; replace L49 with A, G, I, S, T, M or V; replace A50 with G, I, L, S, T, M or V; use G, I , L, S, T, M or V to replace A51; use A, G, I, L, S, M or V to replace T52; use A, G, I, S, T, M or V to replace L53; use A, G, I, S, T, M or V for L54; A, G, I, S, T, M or V for L55; G, I, L, S, T, M or V for A56; A , G, I, S, T, M or V for L57; A, G, I, S, T, M or V for L58; A, G, I, L, T, M or V for S59; A, G, I, L, T, M or V for S60; A, G, I, L, T, M or V for S61; W or Y for F62; A, G, I, L, S , M or V for T63; G, I, L, S, T, M or V for A64; A, G, I, L, S, T or V for M65; A, G, I, L, Replace S66 with T, M or V; replace L67 with A, G, I, S, T, M or V; replace Y68 with F or W; replace Q69 with N; use A, G, I, S, T, M or V for L70; G, I, L, S, T, M or V for A71; G, I, L, S, T, M or V for A72; A, G, I, S, T, M Or replace L73 with V; replace Q74 with N; replace A75 with G, I, L, S, T, M or V; replace D76 with E; replace L77 with A, G, I, S, T, M or V; Replace M78 with A, G, I, L, S, T or V; replace N79 with Q; replace L80 with A, G, I, S, T, M or V; replace R81 with H or K; I, L, S, T or V for M82; D for E83; A, G, I, S, T, M or V for L84; N for Q85; A, G, I, L, T, Replace S86 with M or V; replace Y87 with F or W; replace R88 with H or K; replace G89 with A, I, L, S, T, M or V; use A, G, I, L, T, M or Replace S90 with V; replace A91 with G, I, L, S, T, M or V; replace T92 with A, G, I, L, S, M or V; replace G, I, L, S, T, M or V for A94; G, I, L, S, T, M or V for A95; G, I, L, S, T, M or V for A96; A, I, L, S, T, Replace G97 with M or V; replace A98 with G, I, L, S, T, M or V; replace E100 with D; replace L101 with A, G, I, S, T, M or V; use A, G, Replace T102 with I, L, S, M or V; replace A103 with G, I, L, S, T, M or V; replace G104 with A, I, L, S, T, M or V; replace A, G , I, L, S, T or M for V105; H or R for K106; A, G, I, S, T, M or V for L107; A, G, I, S, T, M or Replace L108 with V; replace T109 with A, G, I, L, S, M or V; replace A111 with G, I, L, S, T, M or V; replace G, I, L, S, T, M or V to replace A112; use H or K to replace R114; use K or R to replace H116; use Q to replace N117; use A, G, I, L, T, M or V to replace S118; use A, G, I, L, Replace S119 with T, M or V; replace R120 with H or K; replace G121 with A, I, L, S, T, M or V; replace H122 with K or R; replace R123 with H or K; replace N124 with Q ; Replace R125 with H or K; Replace R126 with H or K; Replace A127 with G, I, L, S, T, M or V; Replace F128 with W or Y; Replace Q129 with N; Use A, I, L , S, T, M or V for G130; D for E132; D for E133; A, G, I, L, S, M or V for T134; D for E135; N for Q136; E Replace D137; replace V138 with A, G, I, L, S, T or M; replace E139 with D; replace L140 with A, G, I, S, T, M or V; use A, G, I, L , T, M or V for S141; G, I, L, S, T, M or V for A142; G, I, L, S, T, M or V for A145; A, G, I, S, T, M or V for L148; A, I, L, S, T, M or V for G150; H or K for R152; K or R for H153; A, G, I, L, Replace S154 with T, M or V; replace Q155 with N; replace H156 with K or R; replace D157 with E; replace D158 with E; replace N159 with Q; replace with A, I, L, S, T, M or V G160; replace M161 with A, G, I, L, S, T or V; replace N162 with Q; replace L163 with A, G, I, S, T, M or V; replace R164 with H or K; Replace N165; replace I166 with A, G, L; S, T, M or V; replace I167 with A, G, L; S, T, M or V; replace Q168 with N; replace D169 with E; G, I, S, T, M or V for L171; N for Q172; A, G, I, S, T, M or V for L173; A, G, L; S, T, M or V Replace I174; Replace A175 with G, I, L, S, T, M or V; Replace D176 with E; Replace S177 with A, G, I, L, T, M or V; Replace D178 with E; Replace D178 with A , G, I, L, S, M or V for T179; G, I, L, S, T, M or V for A181; A, G, I, S, T, M or V for L182; Replace E183 with D; replace E184 with D; replace K185 with H or R; replace E186 with D; replace N187 with Q; replace K188 with H or R; replace I189 with A, G, L, S, T, M or V; Replace V190 with A, G, I, L, S, T or M; replace V191 with A, G, I, L, S, T or M; replace R192 with H or K; replace Q193 with N; use A, G , I, L, S, M or V for T194; A, I, L, S, T, M or V for G195; F or W for Y196; W or Y for F197; W or Y for F198 ; replace I199 with A, G, L; S, T, M or V; replace Y200 with F or W; replace S201 with A, G, I, L, T, M or V; replace Q202 with N; G, I, L, S, T or M for V203; A, G, I, S, T, M or V for L204; F or W for Y205; A, G, I, L, S, M Or replace T206 with V; replace D207 with E; replace I209 with A, G, L; S, T, M or V; replace F210 with W or Y; replace A211 with G, I, L, S, T, M or V ; Replace M212 with A, G, I, L, S, T or V; Replace G213 with A, I, L, S, T, M or V; Replace H214 with K or R; Use A, G, I, L , S, T or M for V215; A, G, L; S, T, M or V for I216; N for Q217; H or K for R218; H or R for K219; H or R for K220; replace V221 with A, G, I, L, S, T or M; replace H222 with K or R; replace V223 with A, G, I, L, S, T or M; replace F224 with W or Y; Replace G225 with A, I, L, S, T, M or V; replace D226 with E; replace E227 with D; replace L228 with A, G, I, S, T, M or V; , L, T, M or V for S229; A, G, I, S, T, M or V for L230; A, G, I, L, S, T or M for V231; A, G, I, L, S, M or V for T232; A, G, I, S, T, M or V for L233; W or Y for F234; H or K for R235; A, G, L, Replace I237 with S, T, M or V; replace Q238 with N; replace N239 with Q; replace M240 with A, G, I, L, S, T or V; replace K242 with H or R; use A, G, I , L, S, M or V for T243; A, G, I, S, T, M or V for L244; Q for N246; Q for N247; A, G, I, L, T, M or V to replace S248; use F or W to replace Y250; use A, G, I, L, T, M or V to replace S251; use G, I, L, S, T, M or V to replace A252; use A, I , L, S, T, M or V for G253; A, G, L, S, T, M or V for I254; G, I, L, S, T, M or V for A255; H or Replace R256 with K; replace L257 with A, G, I, S, T, M or V; replace E258 with D; replace E259 with D; replace G260 with A, I, L, S, T, M or V; Replace D261; replace E262 with D; replace I263 with A, G, L, S, T, M or V; replace Q264 with N; replace L265 with A, G, I, S, T, M or V; use G, Replace A266 with I, L, S, T, M or V; replace I267 with A, G, L, S, T, M or V; replace R269 with H or K; replace E270 with D; replace N271 with Q; , I, L, S, T, M or V for A272; N for Q273; A, G, L, S, T, M or V for I274; A, G, I, L, T, M or Replace S275 with V; replace R276 with H or K; replace N277 with Q; replace G278 with A, I, L, S, T, M or V; replace D279 with E; replace D280 with E; use A, G, I, Replace T281 with L, S, M or V; replace F282 with W or Y; replace F283 with W or Y; replace G284 with A, I, L, S, T, M or V; use G, I, L, S, T, M or V for A285; A, G, I, S, T, M or V for L286; H or R for K287; A, G, I, S, T, M or V for L288; and /or replace L289 with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (such as enhanced or decreased stability and/or solubility). Preferably, the resulting protein of the invention has increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one increased and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical property.

Amino acids in the Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunnirgham and Wells, Science 244: 1081-1085 (1989)). The latter approach involves a single alanine mutation at every residue in the molecule. The resulting mutants are then tested for functional activity, such as the ability to bind ligand and stimulate lymphocyte (eg, B cell) proliferation, differentiation and/or activation.

Of particular interest is the substitution of charged amino acids with other charged or neutral amino acids, which can result in proteins with desired improved characteristics, such as low aggregation. Aggregation not only reduces activity, but is also a problem when preparing pharmaceutical formulations because aggregates can be immunogenic (Pinckard et al., Clin. Experimental Immunology 2:331-340 (1967); Robbins et al., Diabetes 36:838- 845 (1987); Cleland et al., Crit. Rev. Therepeutic Drug Carrien Systems 10: 307-377 (1993)).

In another embodiment, the present invention provides a polypeptide having an amino acid sequence containing non-conservative substitutions of the amino acid sequence shown in SEQ ID NO:2. For example, non-conservative substitutions of the Neutrokine-α protein sequence shown in SEQ ID NO: 2 include: replacing M1 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing M1 with H, K , R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D2; with H, K, R, A, G, I, L, Replace D3 with S, T, M, V, N, Q, F, W, Y, P, or C; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C S4; replace T5 with D, E, H, K, R, N, Q, F, W, Y, P or C; use H, K, R, A, G, I, L, S, T, M, Replace E6 with V, N, Q, F, W, Y, P, or C; replace E6 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R7; use H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E8; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C replace Q9; use D, E, H, K, R, N , Q, F, W, Y, P, or C to replace S10; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R11; D, E, H, K, R, N, Q, F, W, Y, P, or C for L12; D, E, H, K, R, N, Q, F, W , Y, P, or C for T13; D, E, H, K, R, N, Q, F, W, Y, P, or C for S14; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C15; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L16; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K17; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C replace K18; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R19; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E20; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E21 ; replace M22 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K23; D, E, H, K, R, N, Q, F, W, Y, P, or C for L24; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C replace K25; use H, K, R, A, G, I, L, S, Replace E26 with T, M, V, N, Q, F, W, Y, P, or C; replace E26 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C27; D, E, H, K, R, N, Q, F, W, Y, P, or C for V28; D, E, H, Replace S29 with K, R, N, Q, F, W, Y, P, or C; replace I30 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L31; with D, E, H, K, R, A, G, I, L, S, T, Replace P32 with M, V, N, Q, F, W, Y, or C; replace P32 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R33; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K34; use H, K, Replace E35 with R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace E35 with D, E, H, K, R, N, Q, Replace S36 with F, W, Y, P, or C; replace S36 with D, E, H, K, R, , A, G, I, L, , ST, M, V, N, Q, F, W, Y, or C to replace P37; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S38; use D, E, H, K, R, N, Q, F, Replace V39 with W, Y, P, or C; replace R40 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S41; with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S42; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K43; use H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D44; D, E, H, K, R, N, Q, F, W , Y, P, or C for G45; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K46; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace L47; with D, E, H, K, R, N, Q, F, W, Y, P, or C for L48; D, E, H, K, R, N, Q, F, W, Y, P, or C for A49; D, E, H, K, R, N, Q, F, W , Y, P, or C for A50; D, E, H, K, R, N, Q, F, W, Y, P, or C for T51; D, E, H, K, R, N, Q, F, W, Y, P, or C for L52; D, E, H, K, R, N, Q, F, W, Y, P, or C for L53; D, E, H, Replace L54 with K, R, N, Q, F, W, Y, P, or C; replace A55 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L56; with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L57; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S58; use D, E, H, K, R, A, G, I, Replace C59 with L, S, T, M, V, N, Q, F, W, Y, or P; replace C59 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C60; D, E, H, K, R, N, Q, F, W, Y, P, or C for L61; D, E, Replace T62 with H, K, R, N, Q, F, W, Y, P, or C; replace V63 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Replace V64 with D, E, H, K, R, N, Q, F, W, Y, P, or C; with D, E, H, K, R, N, Q, F, W, Y, P , or C to replace S65; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F66; use D, E, H , K, R, N, Q, A, G, I, L, S, T, M, V, P or C to replace Y67; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C for Q68; D, E, H, K, R, N, Q, F, W, Y, P, or C for V69; Replace A70 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P , or C for A71; D, E, H, K, R, N, Q, F, W, Y, P, or C for L72; D, E, H, K, R, A, G, I , L, S, T, M, V, F, , W, Y, P, or C to replace Q73; use D, E, H, K, R, N, Q, F, W, Y, P, or C Replace G74; replace D75 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace D, E, H, K , R, N, Q, F, W, Y, P, or C for L76; D, E, H, K, R, N, Q, F, W, Y, P, or C for A77; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace S78; with D, E, H, K, R, N, Q, F, W, Y, P, or Replace L79 with C; replace R80 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace D, E, H, K , R, N, Q, F, W, Y, P, or C to replace A81; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W , Y, P, or C for E82; D, E, H, K, R, N, Q, F, W, Y, P, or C for L83; D, E, H, K, R, A , G, I, L, S, T, M, V, F, , W, Y, P, or C to replace Q84; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G85; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace H86; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace H87; with D, E, H, K, R, N, Q, F, W, Y, P, or C for A88; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E89 ; replace K90 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R, Replace L91 with N, Q, F, W, Y, P, or C; replace L91 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P92; D, E, H, K, R, N, Q, F, W, Y, P, or C for A93; D, E, H, K, R, N, Q, F, W, Y, P, or C for G94; D, E, H, K, R, N, Q, F, W, Y, P, or C for A95; D, E, H, K, R, N, Q, F, W, Y, P, or C for G96; D, E, H, K, R, N, Q, F, W, Y, P, or C for A97; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P98; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C replace K99; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A100; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G101; use D, E, H, K, R, N, Q, Replace L102 with F, W, Y, P, or C; replace L102 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace E103; replace E104 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace D, E, H, Replace A105 with K, R, N, Q, F, W, Y, P, or C; replace A105 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P106; D, E, H, K, R, N, Q, F, W, Y, P, or C for A107; D, E, H, K, R, N, Q, F, W, Y, P, or C for V108; D, E, H, K, R, N, Q, F, W, Y, P, or C for T109; D, E , H, K, R, N, Q, F, W, Y, P, or C to replace A110; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C G111; replace L112 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T, M, V , N, Q, F, W, Y, P, or C for K113; D, E, H, K, R, N, Q, F, W, Y, P, or C for I114; D, E , H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C to replace F115; with H, K, R, A, G, I, L, S, Replace E116 with T, M, V, N, Q, F, W, Y, P, or C; replace E116 with D, E, H, K, R, A, G, I, L, S, T, M, V, Replace P117 with N, Q, F, W, Y, or C; replace P117 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Replace P118 with Y, or C; replace A119 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace A119 with D, E, H, K, R, A, G, Replace P120 with I, L, S, T, M, V, N, Q, F, W, Y, or C; replace P120 with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G121; replace E122 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, Replace G123 with H, K, R, N, Q, F, W, Y, P, or C; replace G123 with D, E, H, K, R, A, G, I, L, S, T, M, V, Replace N124 with F, W, Y, P or C; replace S125 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R , N, Q, F, W, Y, P, or C to replace S126; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C for Q127; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for N128; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace S129; with D, E, A, G, I, L, S, T, M, V, N, Q , F, W, Y, P, or C for R130; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C Substitute N131; Substitute D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K132; Substitute D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R133; with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A134; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace V135; use D, E, H, K, R, A, G, Replace Q136 with I, L, S, T, M, V, F, , W, Y, P, or C; replace Q136 with D, E, H, K, R, N, Q, F, W, Y, P, or C for G137; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P138; H, K , R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E139; with H, K, R, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P, or C to replace E140; with D, E, H, K, R, N, Q, F, W, Y, P, or C Replace T141; replace V142 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace T143 with Y, P or C; replace Q144 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C replace D145; use D, E, H, K, R, A , G, I, L, S, T, M, V, N, Q, F, W, Y, or P to replace C146; with D, E, H, K, R, N, Q, F, W, Y , P, or C for L147; D, E, H, K, R, A, G, I, L, S, T, M, V, F,, W, Y, P, or C for Q148; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L149; with D, E, H, K, R, N, Q, F, W, Y, P, Or C to replace I150; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A151; use H, K, R, A, G, I, L, S, Replace D152 with T, M, V, N, Q, F, W, Y, P or C; replace S153 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Replace E154 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R , N, Q, F, W, Y, P or C to replace T155; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P156; D, E, H, K, R, N, Q, F, W, Y, P, or C for T157; D, E, H, K, R, N, Q , F, W, Y, P, or C to replace I158; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C to replace Q159; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K160; use D, E, H, K, R, N, Q, F, W, Y, P, or C for G161; D, E, H, K, R, N, Q, F, W, Y, P, or C for S162; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C to replace Y163; use D, E, H, K, R, N, Q , F, W, Y, P or C for T164; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C for F165; Replace V166 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, A, G, I, L, S, T , M, V, N, Q, F, W, Y, or C to replace P167; with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C Replace W168; replace L169 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace L170 with Y, P, or C; replace S171 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Replace F172 with Q, A, G, I, L, S, T, M, V, P or C; replace F172 with D, E, A, G, I, L, S, T, M, V, N, Q, F , W, Y, P, or C to replace K173; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R174; Replace G175 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P , or C for S176; D, E, H, K, R, N, Q, F, W, Y, P, or C for A177; D, E, H, K, R, N, Q, F , W, Y, P, or C to replace L178; replace with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C E179; Substitution of E180 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Substitution of D, E, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K181; H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P, or C to replace E182; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W , Y, P or C to replace N183; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K184; use D, E, H, K, R, N, Q, F, W, Y, P, or C for I185; D, E, H, K, R, N, Q, F, W, Y, P, or C Replace L186; replace V187 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T, M, Replace K188 with V, N, Q, F, W, Y, P, or C; replace K188 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Replace E189 with Y, P, or C; Replace T190 with D, E, H, K, R, N, Q, F, W, Y, P or C; Replace D, E, H, K, R, N, Replace G191 with Q, F, W, Y, P, or C; replace with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C Y192; replace F193 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replace D, E, H, K, R, Replace F194 with N, Q, A, G, I, L, S, T, M, V, P or C; replace F194 with D, E, H, K, R, N, Q, F, W, Y, P, or C for I195; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C for Y196; D, E, H, K, Replace G197 with R, N, Q, F, W, Y, P, or C; replace G197 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W , Y, P, or C for Q198; D, E, H, K, R, N, Q, F, W, Y, P, or C for V199; D, E, H, K, R, N , Q, F, W, Y, P, or C to replace L200; with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C Substitute Y201; Substitute T202 with D, E, H, K, R, N, Q, F, W, Y, P or C; Substitute H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P or C to replace D203; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K204; use D, E, H, K, R, N, Q, F, W, Y, P or C to replace T205; use D, E, H, K, R, N, Q, A , G, I, L, S, T, M, V, P or C for Y206; D, E, H, K, R, N, Q, F, W, Y, P, or C for A207; Replace M208 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace M208 with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G209; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace H210; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L211; D, E, H, K, R, N, Q, F, W, Y, P, or C for I212; D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C replace Q213; use D, E, A, G, I , L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R214; with D, E, A, G, I, L, S, T, M, V, N , Q, F, W, Y, P, or C to replace K215; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K216; D, E, H, K, R, N, Q, F, W, Y, P, or C for V217; D, E, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P, or C for H218; D, E, H, K, R, N, Q, F, W, Y, P, or C for V219; D , E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C to replace F220; with D, E, H, K, R, N, Q, Replace G221 with F, W, Y, P, or C; replace with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C D222; Substitution of E223 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Substitution of D, E, H, K , R, N, Q, F, W, Y, P, or C for L224; D, E, H, K, R, N, Q, F, W, Y, P, or C for S225; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace L226; with D, E, H, K, R, N, Q, F, W, Y, P, or C for V227; D, E, H, K, R, N, Q, F, W, Y, P or C for T228; D, E, H, K, R, N, Q, F, W, Replace L229 with Y, P, or C; replace F230 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replace D, E , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R231; use D, E, H, K, R, A, G, I , L, S, T, M, V, N, Q, F, W, Y, or P to replace C232; with D, E, H, K, R, N, Q, F, W, Y, P, or Replace I233 with C; replace Q234 with D, E, H, K, R, A, G, I, L, S, T, M, V, F,, W, Y, P, or C; replace D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for N235; D, E, H, K, R, N, Q, F , W, Y, P, or C to replace M236; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P237; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E238; D, E, H , K, R, N, Q, F, W, Y, P or C for T239; D, E, H, K, R, N, Q, F, W, Y, P, or C for L240; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P241; with D, E, H, K, Replace N242 with R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replace N242 with D, E, H, K, R, A, G, I, L, S , T, M, V, F, W, Y, P or C to replace N243; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S244; use D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P to replace C245; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for Y246; D, E, H, K, R, N, Q, F, W, Y, P, or C for S247; D, E, H, K, R, N, Q, F, W, Y, P, or C for A248; D, E, H, K, R, N, Q, F, W , Y, P, or C for G249; D, E, H, K, R, N, Q, F, W, Y, P, or C for I250; D, E, H, K, R, N , Q, F, W, Y, P, or C to replace A251; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K252; D, E, H, K, R, N, Q, F, W, Y, P, or C for L253; H, K, R, A, G, I, L, S, T , M, V, N, Q, F, W, Y, P, or C to replace E254; H, K, R, A, G, I, L, S, T, M, V, N, Q, F , W, Y, P, or C for E255; D, E, H, K, R, N, Q, F, W, Y, P, or C for G256; H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D257; with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E258; D, E, H, K, R, N, Q, F, W, Y, P, or C for L259; D, E, H, K, R, N, Q, F, W, Y, P, or C for L259; E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C replace Q260; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L261; D, E, H, K, R, N, Q, F, W, Y, P, or C for A262; D, E, H, Replace I263 with K, R, N, Q, F, W, Y, P, or C; replace I263 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P264; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R265 ; replace E266 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for N267; D, E, H, K, R, N, Q, F, W, Y , P, or C for A268; D, E, H, K, R, A, G, I, L, S, T, M, V, F,, W, Y, P, or C for Q269; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I270; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S271; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L272; use H, K, R, A, G, I, L, S, Replace D273 with T, M, V, N, Q, F, W, Y, P, or C; replace G274 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Replace D275 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use D, E, H, K, R, Replace T276 with N, Q, F, W, Y, P or C; replace T277 with D, E, H, K, R, N, Q, F, W, Y, P or C; replace D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C for F278; D, E, H, K, R, N, Q, A, G, I , L, S, T, M, V, P or C to replace F279; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G280; use D, E, H, K, R, N, Q, F, W, Y, P, or C for A281; D, E, H, K, R, N, Q, F, W, Y, P, or C for L282 ; replace K283 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L284; and/or D, E, H, K, R, N, Q, F, W, Y, P, or C for L285. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The obtained Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (such as enhanced or reduced stability and/or solubility) described herein and known in the art. ). Preferably, the resulting protein of the invention has enhanced and/or reduced Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the invention has more than one increased and/or decreased functional activity and/or physical property of Neutrokine-α and/or Neutrokine-αSV.

In another embodiment, the Neutrokine-α polypeptide of the present invention comprises more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) of the above amino acids Substituted amino acids (conserved or non-conserved).

In another embodiment of the present invention, the non-conservative substitutions in the Neutrokine-αSV protein sequence shown in SEQ ID NO: 19 include: using D, E, H, K, R, N, Q, F, W, Y, Replace M1 with P, or C; replace D2 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use H, K , R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D3; with D, E, H, K, R, N, Q, F, W, Y, P, or C for S4; D, E, H, K, R, N, Q, F, W, Y, P, or C for T5; H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E6; with D, E, A, G, I, L, S, T, M, V , N, Q, F, W, Y, P, or C to replace R7; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y , P, or C to replace E8; use D, E, H, K, R, A, G, I, L, S, T, M, V, W, Y, P, or C to replace Q9; use D, E , H, K, R, N, Q, F, W, Y, P, or C to replace S10; replace S10 with D, E, A, G, I, L, S, T, M, V, N, Q, F , W, Y, P, or C for R11; D, E, H, K, R, N, Q, F, W, Y, P, or C for L12; D, E, H, K, R , N, Q, F, W, Y, P, or C for T13; D, E, H, K, R, N, Q, F, W, Y, P, or C for S14; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C15; D, E, H, K, R, N, Replace L16 with Q, F, W, Y, P, or C; replace L16 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace K17; replace K18 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R19; H, K, R, A, G, I, L, S, T, M, Replace E20 with V, N, Q, F, W, Y, P, or C; replace E20 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E21; D, E, H, K, R, N, Q, F, W, Y, P, or C for M22; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K23; D, E, H, K, R, N, Q, F, W, Y, P, or C Replace L24; replace K25 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace H, K, R, A, Replace E26 with G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace E26 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C27; D, E, H, K, R, N, Q, F, W, Y, P, or C for V28 ; replace S29 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I30; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L31; use D, E, H, K, R, A, G, Replace P32 with I, L, S, T, M, V, N, Q, F, W, Y, or C; replace P32 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R33; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace K34; replace E35 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace D, E, H, Replace S36 with K, R, N, Q, F, W, Y, P, or C; replace S36 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P37; D, E, H, K, R, N, Q, F, W, Y, P, or C for S38; D, E, H, K, Replace V39 with R, N, Q, F, W, Y, P, or C; replace V39 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R40; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S41; use D, E, H, K, R, N, Q, Replace S42 with F, W, Y, P, or C; replace K43 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C ; replace D44 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use D, E, H, K, R , N, Q, F, W, Y, P, or C to replace G45; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P , or C for K46; D, E, H, K, R, N, Q, F, W, Y, P, or C for L47; D, E, H, K, R, N, Q, F , W, Y, P, or C for L48; D, E, H, K, R, N, Q, F, W, Y, P, or C for A49; D, E, H, K, R , N, Q, F, W, Y, P, or C to replace A50; use D, E, H, K, R, N, Q, F, W, Y, P or C to replace T51; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L52; D, E, H, K, R, N, Q, F, W, Y, P, or C for L53 ; Replace L54 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Substitute D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A55; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L56; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L57; D, E, H, K, R, N, Q, F, W, Y, P, or C for S58; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C59; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C60; D, E, H, K, R, N, Q, F, W, Y, P, or C Replace L61; replace T62 with D, E, H, K, R, N, Q, F, W, Y, P or C; replace D, E, H, K, R, N, Q, F, W, Y , P, or C for V63; D, E, H, K, R, N, Q, F, W, Y, P, or C for V64; D, E, H, K, R, N, Q , F, W, Y, P, or C to replace S65; to replace S65 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C F66; replace Y67 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C for Q68; D, E, H, K, R, N, Q, F, W, Y , P, or C to replace V69; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A70; use D, E, H, K, R, N, Q , F, W, Y, P, or C for A71; D, E, H, K, R, N, Q, F, W, Y, P, or C for L72; D, E, H, K , R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C to replace Q73; use D, E, H, K, R, N, Q, F, W, Y, P, or C for G74; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for D75; Replace L76 with D, E, H, K, R, N, Q, F, W, Y, P, or C; with D, E, H, K, R, N, Q, F, W, Y, P , or C for A77; D, E, H, K, R, N, Q, F, W, Y, P, or C for S78; D, E, H, K, R, N, Q, F , W, Y, P, or C to replace L79; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R80; Replace A81 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use H, K, R, A, G, I, L, S, T, M, V , N, Q, F, W, Y, P, or C for E82; D, E, H, K, R, N, Q, F, W, Y, P, or C for L83; D, E , H, K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C to replace Q84; use D, E, H, K, R, N, Replace G85 with Q, F, W, Y, P, or C; replace G85 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace H86; replace H87 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace D, E, H, K, Replace A88 with R, N, Q, F, W, Y, P, or C; replace A88 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E89; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K90; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L91; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P92; D, E, H, K, R, N, Q, F, W, Y, P, or C for A93; D, E, H, K, R, N, Q, F, W, Y, P, or C for G94; D, E, H, K, R, N, Q, F, W, Y, P, or C for A95 ; replace G96 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y, Replace A97 with P, or C; replace P98 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C replace K99; use D, E, H, K, R, N, Q, F, W, Y, P, or C for A100; D, E, H, K, R, N, Q, F, W, Y, P, or C for G101; D, E, H, Replace L102 with K, R, N, Q, F, W, Y, P, or C; replace L102 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E103; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E104 ; replace A105 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, A, G, I, L, S, Replace P106 with T, M, V, N, Q, F, W, Y, or C; replace A107 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace V108; use D, E, H, K, R, N, Q, F, W, Y, P or C for T109; D, E, H, K, R, N, Q, F, W, Y, P, or C for A110; D, E, H, K, R, N, Q, F, W , Y, P, or C for G111; D, E, H, K, R, N, Q, F, W, Y, P, or C for L112; D, E, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P, or C to replace K113; with D, E, H, K, R, N, Q, F, W, Y, P, or C for I114; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C for F115; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E116; with D, E, H, K, R, A, G, I, L, Replace P117 with S, T, M, V, N, Q, F, W, Y, or C; replace P117 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P118; D, E, H, K, R, N, Q, F, W, Y, P, or C for A119; D, E, H, Replace P120 with K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replace D, E, H, K, R, N, Q, Replace G121 with F, W, Y, P, or C; replace G121 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace E122; replace G123 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for N124; D, E, H, K, R, N, Q, F, W, Y, P, or C for S125; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace S126; with D, E, H, K, R, A, G, I, L, S, T, M , V, F, , W, Y, P, or C to replace Q127; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, Replace N128 with P or C; replace S129 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T , M, V, N, Q, F, W, Y, P, or C to replace R130; with D, E, H, K, R, A, G, I, L, S, T, M, V, F , W, Y, P or C to replace N131; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K132; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R133; with D, E, H, K, R, N, Q, F, W, Y, P, or C for A134; D, E, H, K, R, N, Q, F, W, Y, P, or C for V135; D, E, H, Replace Q136 with K, R, A, G, I, L, S, T, M, V, F, , W, Y, P, or C; use D, E, H, K, R, N, Q, F , W, Y, P, or C for G137; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or Replace P138 with C; replace E139 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace E139 with H, K, R , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E140; use D, E, H, K, R, N, Q, F , W, Y, P, or C for T141; D, E, H, K, R, N, Q, F, W, Y, P, or C for G142; D, E, H, K, R, Replace S143 with N, Q, F, W, Y, P, or C; replace Y144 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C ; Replace T145 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Substitute D, E, H, K, R, N, Q, A, G, I, Replace F146 with L, S, T, M, V, P, or C; replace V147 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H , K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P148; with D, E, H, K, R, N, Q , A, G, I, L, S, T, M, V, P, or C for W149; D, E, H, K, R, N, Q, F, W, Y, P, or C for L150 ; Replace L151 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Substitute D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S152; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F153; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C replace K154; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R155; D, E, H, K, R, N, Q, F, W, Y, P, or C for G156; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S157; with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A158; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L159; use H, K, R, A, G, I, L, S, Replace E160 with T, M, V, N, Q, F, W, Y, P, or C; replace E160 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E161; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K162 ; replace E163 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, Replace N164 with R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replace N164 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K165; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I166; use D, Replace L167 with E, H, K, R, N, Q, F, W, Y, P, or C; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C V168; replace K169 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E170; with D, E, H, K, R, N, Q, F, W, Y , P, or C for T171; D, E, H, K, R, N, Q, F, W, Y, P, or C for G172; D, E, H, K, R, A, G , I, L, S, T, M, V, P, or C to replace Y173; with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V , P, or C for F174; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for F175; D, E , H, K, R, N, Q, F, W, Y, P, or C to replace I176; with D, E, H, K, R, A, G, I, L, S, T, M, V , P, or C for Y177; D, E, H, K, R, N, Q, F, W, Y, P, or C for G178; D, E, H, K, R, A, G , I, L, S, T, M, V, M, V, P, or C for Q179; D, E, H, K, R, N, Q, F, W, Y, P, or C for V180 ; Replace L181 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Substitute D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C for Y182; D, E, H, K, R, N, Q, F, W, Y, P, or C for T183; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D184; with D, E, A, G, I, L, S, T, M, V , N, Q, F, W, Y, P, or C for K185; D, E, H, K, R, N, Q, F, W, Y, P, or C for T186; D, E , H, K, R, A, G, I, L, S, T, M, V, P, or C for Y187; D, E, H, K, R, N, Q, F, W, Y , P, or C for A188; D, E, H, K, R, N, Q, F, W, Y, P, or C for M189; D, E, H, K, R, N, Q , F, W, Y, P, or C to replace G190; to replace G190 with D, E, , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C H191; replace L192 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Y , P, or C for I193; D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C for Q194; D, E , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R195; use D, E, A, G, I, L, S, T , M, V, N, Q, F, W, Y, P, or C to replace K196; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W , Y, P, or C for K197; D, E, H, K, R, N, Q, F, W, Y, P, or C for V198; D, E,, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P or C to replace H199; replace with D, E, H, K, R, N, Q, F, W, Y, P or C V200; replace F201 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; use D, E, H, K, R , N, Q, F, W, Y, P, or C to replace G202; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y , P or C to replace D203; use H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E204; use D, Replace L205 with E, H, K, R, N, Q, F, W, Y, P, or C; replace L205 with D, E, H, K, R, N, Q, F, W, Y, P, or C Replace S206; replace L207 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Y, P or C for V208; D, E, H, K, R, N, Q, F, W, Y, P, or C for T209; D, E, H, K, R, N, Q , F, W, Y, P, or C to replace L210; replace with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C F211; replace R212 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R , A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C213; D, E, H, K, R, N, Q, F, W , Y, P, or C to replace I214; use D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C to replace Q215; use D , E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N216; use D, E, H, K, R, N , Q, F, W, Y, P, or C to replace M217; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W , Y, or C for P218; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E219; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace T220; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L221; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C for P222; D, E , H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N223; use D, E, H, K, R, A, G , I, L, S, T, M, V, F, W, Y, P, or C for N224; D, E, H, K, R, N, Q, F, W, Y, P, or Replace S225 with C; replace C226 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replace D, E , H, K, R, A, G, I, L, S, T, M, V, P, or C for Y227; D, E, H, K, R, N, Q, F, W, Y , P, or C for S228; D, E, H, K, R, N, Q, F, W, Y, P, or C for A229; D, E, H, K, R, N, Q , F, W, Y, P, or C for G230; D, E, H, K, R, N, Q, F, W, Y, P, or C for I231; D, E, H, K , R, N, Q, F, W, Y, P, or C to replace A232; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y , P, or C for K233; D, E, H, K, R, N, Q, F, W, Y, P, or C for L234; H, K, R, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P, or C to replace E235; H, K, R, A, G, I, L, S, T, M, V, N , Q, F, W, Y, P, or C for E236; D, E, H, K, R, N, Q, F, W, Y, P, or C for G237; H, K, R , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D238; with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for E239; D, E, H, K, R, N, Q, F, W, Y, P, or C for L240 ; replace Q241 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L242; D, E, H, K, R, N, Q, F, W, Y, P, or C for A243; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I244; with D, E, H, K, R, A, G, I, L, S, T, M, V, Replace P245 with N, Q, F, W, Y, or C; replace P245 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace R246; replace E247 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for N248; D, E, H, K, R, N, Q, F, Replace A249 with W, Y, P, or C; replace Q250 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I251; with D, E, H, K, R, N, Q, F, W, Y, P, Or C to replace S252; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L253; use H, K, R, A, G, I, L, S, Replace D254 with T, M, V, N, Q, F, W, Y, P or C; replace G255 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Replace D256 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use D, E, H, K, R, N, Q, F, W, Y, P, or C for V257; D, E, H, K, R, N, Q, F, W, Y, P, or C for T258; D, E, H , K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F259; use D, E, H, K, R, N, Q, A, G , I, L, S, T, M, V, P, or C to replace F260; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G261; use D , E, H, K, R, N, Q, F, W, Y, P, or C to replace A262; with D, E, H, K, R, N, Q, F, W, Y, P, or C for L263; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K264; D, E, H, K , R, N, Q, F, W, Y, P, or C for L265; and/or D, E, H, K, R, N, Q, F, W, Y, P, or C for L266 . Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (such as enhanced or reduced stability and/or solubility) as described herein and known in the art. ). Preferably, the resulting protein of the invention has enhanced and/or decreased Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one enhanced and/or decreased functional activity and/or physical property of Neutrokine-α and/or Neutrokine-αSV.

In another embodiment, the Neutrokine-α polypeptide of the present invention comprises more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids.

For example, preferred non-conservative substitutions of the Neutrokine-alpha protein sequence shown in SEQ ID NO: 23 include: with D, E, A, G, I, L, S, T, M, V, N, Q, F, W , Y, P, or C for R1; D, E, H, K, R, N, Q, F, W, Y, P, or C for V2; D, E, H, K, R, N, Replace V3 with Q, F, W, Y, P or C; replace V3 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C Replace D4; replace L5 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Y, P, or C for S6; D, E, H, K, R, N, Q, F, W, Y, P, or C for A7; D, E, H, K, R, A, Replace P8 with G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replace P8 with D, E, H, K, R, A, G, I, L, S, Replace P9 with T, M, V, N, Q, F, W, Y, or C; replace A10 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P11; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P for C12; D, E, H, K, R, N, Q, F, Replace L13 with W, Y, P, or C; replace L13 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C Replace P14; replace G15 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, A, G, I, L, Replace C16 with S, T, M, V, N, Q, F, W, Y, or P; replace C16 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R17; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for H18; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace S19; with D, E, H, K, R, A, G, I, L, S, T, M , V, F, W, Y, P, or C to replace Q20; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace H21; replace D22 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace H, K, R, A , G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D23; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for N24; D, E, H, K, R, N, Q, F, W, Y, P, or C for G25; D , E, H, K, R, N, Q, F, W, Y, P or C to replace M26; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for N27; D, E, H, K, R, N, Q, F, W, Y, P, or C for L28; D, E, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R29; with D, E, H, K, R, A, G, I, L, S , T, M, V, F, W, Y, P or C to replace N30; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R31; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace T32; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for Y33; D, E, H, K, R, N, Q, F, W, Y, P, or C for T34 ; replace F35 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; use D, E, H, K, R, Replace V36 with N, Q, F, W, Y, P or C; replace V36 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W , Y, or C for P37; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for W38; D, E , H, K, R, N, Q, F, W, Y, P, or C to replace L39; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C L40; replace S41 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, A, G, I , L, S, T, M, V, P, or C to replace F42; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P , or C to replace K43; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace R44; use D, E, H , K, R, N, Q, F, W, Y, P, or C to replace G45; with D, E, H, K, R, A, G, I, L, S, T, M, V, F , W, Y, P, or C for N46; D, E, H, K, R, N, Q, F, W, Y, P, or C for A47; D, E, H, K, R, Replace L48 with N, Q, F, W, Y, P, or C; replace L48 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E49; use H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E50; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K51; with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E52; with D, E, H, K, R, A, G, I, L, S, T, M, Replace N53 with V, F, W, Y, P or C; replace with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C K54; replace I55 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, F, W, Y , P or C to replace V56; use D, E, H, K, R, N, Q, F, W, Y, P or C to replace V57; use D, E, A, G, I, L, S, T , M, V, N, Q, F, W, Y, P, or C to replace R58; with D, E, H, K, R, A, G, I, L, S, T, M, V, F , W, Y, P, or C for Q59; D, E, H, K, R, N, Q, F, W, Y, P, or C for T60; D, E, H, K, R , N, Q, F, W, Y, P, or C to replace G61; with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P , or C to replace Y62; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F63; use D, E, H , K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F64; use D, E, H, K, R, N, Q, F, W , Y, P, or C to replace I65; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace Y66; use D , E, H, K, R, N, Q, F, W, Y, P, or C to replace S67; with D, E, H, K, R, A, G, I, L, S, T, M , V, F, W, Y, P, or C for Q68; D, E, H, K, R, N, Q, F, W, Y, P, or C for V69; D, E, H, Replace L70 with K, R, N, Q, F, W, Y, P, or C; replace L70 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for Y71; D, E, H, K, R, N, Q, F, W, Y, P, or C for T72; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D73; D, E, H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, or C for P74; D, E, H, K, R, N, Q, F, W, Y, P, or C for I75; D, E , H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F76; use D, E, H, K, R, N, Q, F , W, Y, P, or C for A77; D, E, H, K, R, N, Q, F, W, Y, P, or C for M78; D, E, H, K, R, Replace G79 with N, Q, F, W, Y, P, or C; replace G79 with D, E, A, G, I, L, S, T, M, V, , N, Q, F, W, Y, P , or C for H80; D, E, H, K, R, N, Q, F, W, Y, P or C for V81; D, E, H, K, R, N, Q, F, W, Y, P, or C for I82; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for Q83 ; Replace R84 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Use D, E, A, G, I, Replace K85 with L, S, T, M, V, N, Q, F, W, Y, P, or C; replace K85 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K86; D, E, H, K, R, N, Q, F, W, Y, P, or C for V87; D, E, A, G , I, L, S, T, M, V, , N, Q, F, W, Y, P, or C to replace H88; with D, E, H, K, R, N, Q, F, W, Y, P or C for V89; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for F90; D, E , H, K, R, N, Q, F, W, Y, P, or C to replace G91; replace G91 with H, K, R, A, G, I, L, S, T, M, V, N, Q , F, W, Y, P or C to replace D92; replace D92 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace E93; replace L94 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace S95 with Y, P, or C; replace L96 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Y, P, or C for V97; D, E, H, K, R, N, Q, F, W, Y, P, or C for T98; D, E, H, K , R, N, Q, F, W, Y, P, or C to replace L99; with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V , P, or C for F100; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R101; D, E , H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, replace C102; use D, E, H, K, R, Replace I103 with N, Q, F, W, Y, P, or C; replace I103 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace Q104; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C to replace N105; use D, E , H, K, R, N, Q, F, W, Y, P or C to replace M106; with D, E, H, K, R, A, G, I, L, S, T, M, V, Replace P107 with N, Q, F, W, Y, or C; replace P107 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace K108; replace T109 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace L110 with Y, P, or C; replace P111 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C ; replace N112 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; use D, E, H, K, R , A, G, I, L, S, T, M, V, F, W, Y, P or C to replace N113; with D, E, H, K, R, N, Q, F, W, Y, Replace S114 with P, or C; replace C115 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Replace Y116 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; use D, E, H, K, R, N , Q, F, W, Y, P, or C for S117; D, E, H, K, R, N, Q, F, W, Y, P, or C for A118; D, E, H , K, R, N, Q, F, W, Y, P, or C for G119; D, E, H, K, R, N, Q, F, W, Y, P, or C for I120; Replace A121 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, A, G, I, L, S, T, M, V, N , Q, F, W, Y, P, or C for R122; D, E, H, K, R, N, Q, F, W, Y, P, or C for L123; H, K, R , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace E124; with H, K, R, A, G, I, L, S , T, M, V, N, Q, F, W, Y, P, or C to replace E125; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C G126; replace D127 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use H, K, R, A, Replace E128 with G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace E128 with D, E, H, K, R, N, Q, F, W, Y, P, or C for I129; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for Q130; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L131; with D, E, H, K, R, N, Q, F, W, Y, P, Or C to replace A132; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace I133; use D, E, H, K, R, A, G, I, Replace P134 with L, S, T, M, V, N, Q, F, W, Y, or C; replace P134 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R135; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C Replace E136; replace N137 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replace D, E, H, K , R, N, Q, F, W, Y, P, or C to replace A138; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W , Y, P, or C for Q139; D, E, H, K, R, N, Q, F, W, Y, P, or C for I140; D, E, H, K, R, N , Q, F, W, Y, P, or C to replace S141; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R142; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for N143; D, E, H, Replace G144 with K, R, N, Q, F, W, Y, P, or C; replace G144 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, Replace D145 with W, Y, P or C; replace D146 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace D146 with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace T147; with D, E, H, K, R, N, Q, A, G, I, L, Replace F148 with S, T, M, V, P, or C; replace F148 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C Replace F149; replace G150 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace A151 with Y, P, or C; replace L152 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K153; D, E, H, K, R, N, Q, F, W, Y, P, or C Substitution of L154; and/or substitution of L155 with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha protein of the invention can be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or solubility as described herein and known in the art. Preferably, the resulting protein of the invention has enhanced or decreased Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one enhanced or decreased Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical property.

In another embodiment, the Neutrokine-α polypeptide of the present invention comprises more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids.

For example, preferred non-conservative substitutions for the Neutrokine-α protein sequence shown in SEQ ID NO: 38 include: replacing M1 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing M1 with H , K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D2; with H, K, R, A, G, I, Replace E3 with L, S, T, M, V, N, Q, F, W, Y, P or C; replace E3 with D, E, H, K, R, N, Q, F, W, Y, P, or C for S4; D, E, H, K, R, N, Q, F, W, Y, P, or C for A5; D, E, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P, or C to replace K6; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace T7; use D , E, H, K, R, N, Q, F, W, Y, P, or C to replace L8; use D, E, H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, or C to replace P9; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F , W, Y, or C for P10; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C P11; replace C12 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; use D, E, Replace L13 with H, K, R, N, Q, F, W, Y, P, or C; replace L13 with D, E, H, K, R, A, G, I, L, S, T, M, V, Replace C14 with N, Q, F, W, Y, or P; replace C14 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F15; use D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P to replace C16; use D, Replace S17 with E, H, K, R, N, Q, F, W, Y, P, or C; replace S17 with H, K, R, A, G, I, L, S, T, M, V, N, Substitute E18 with Q, F, W, Y, P or C; Substitute with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C K19; replace G20 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P or C to replace E21; with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Replace D22 with Y, P or C; replace M23 with D, E, H, K, R, N, Q, F, W, Y, P or C; replace D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K24; D, E, H, K, R, N, Q, F, W, Y, P, or C for V25 ; replace G26 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, N, Q, A, G, I, Replace Y27 with L, S, T, M, V, P, or C; replace Y27 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, Replace D28 with P or C; replace P29 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replace D with D , E, H, K, R, N, Q, F, W, Y, P, or C to replace I30; with D, E, H, K, R, N, Q, F, W, Y, P, or Replace T31 with C; replace P32 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replace D, E , H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C to replace Q33; use D, E, A, G, I, L, S, Replace K34 with T, M, V, N, Q, F, W, Y, P, or C; replace K34 with H, K, R, A, G, I, L, S, T, M, V, N, Q, Replace E35 with F, W, Y, P or C; replace E36 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C ; Substitute D, E, H, K, R, N, Q, F, W, Y, P, or C for G37; Substitute D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A38; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace W39; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F40; with D, E, H, K, R, N, Q, F, W, Y, P, or C for G41; D, E, H, K, R, N, Q, F, W, Y, P, or C for I42; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, replacing C43; with D, E, A, G, I, L, S, T, M , V, N, Q, F, W, Y, P or C to replace R44; with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for D45; D, E, H, K, R, N, Q, F, W, Y, P, or C for G46; D, E, A, G, I, L, S , T, M, V, N, Q, F, W, Y, P, or C for R47; D, E, H, K, R, N, Q, F, W, Y, P, or C for L48 ; Replace L49 with D, E, H, K, R, N, Q, F, W, Y, P, or C; Substitute D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A50; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A51; use D, E, H, K, R, N, Q, F, W, Y, P, or C for T52; D, E, H, K, R, N, Q, F, W, Y, P, or C for L53; D, E, H, K, R, N, Q, F, W, Y, P, or C for L54; D, E, H, K, R, N, Q, F, W, Y, P, or C for L55; D, E, H, K, R, N, Q, F, W, Y, P, or C for L55; E, H, K, R, N, Q, F, W, Y, P, or C to replace A56; with D, E, H, K, R, N, Q, F, W, Y, P, or C Replace L57; replace L58 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Replace S59 with Y, P, or C; replace S60 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Replace S61 with Q, F, W, Y, P, or C; replace S61 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C Replace F62; replace T63 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F, W, Y, P, or C for A64; D, E, H, K, R, N, Q, F, W, Y, P, or C for M65; D, E, H, K, R, N, Q , F, W, Y, P, or C for S66; D, E, H, K, R, N, Q, F, W, Y, P, or C for L67; D, E, H, K , R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace Y68; use D, E, H, K, R, A, G, I, L, S , T, M, V, F, W, Y, P or C for Q69; D, E, H, K, R, N, Q, F, W, Y, P, or C for L70; D, E, H, K, R, N, Q, F, W, Y, P, or C for L70; E, H, K, R, N, Q, F, W, Y, P, or C to replace A71; with D, E, H, K, R, N, Q, F, W, Y, P, or C Replace A72; replace L73 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for Q74; D, E, H, K, R, N, Q, F, W, Y, P, or C for A75; H , K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D76; use D, E, H, K, R, N, Q, F, W, Y, P, or C for L77; D, E, H, K, R, N, Q, F, W, Y, P, or C for M78; D, E, H, K , R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N79; use D, E, H, K, R, N, Q, F, W , Y, P, or C to replace L80; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace R81; use D, Replace M82 with E, H, K, R, N, Q, F, W, Y, P or C; replace M82 with H, K, R, A, G, I, L, S, T, M, V, N, Q , F, W, Y, P, or C for E83; D, E, H, K, R, N, Q, F, W, Y, P, or C for L84; D, E, H, K, Replace Q85 with R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replace Q85 with D, E, H, K, R, N, Q, F, W, Y , P, or C for S86; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for Y87; D, E , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace R88; with D, E, H, K, R, N, Q, F, W, Y, P, or C for G89; D, E, H, K, R, N, Q, F, W, Y, P, or C for S90; D, E, H, K, R, N, Q, F, W, Y, P, or C for A91; D, E, H, K, R, N, Q, F, W, Y, P, or C for T92; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P93; with D, E, H, K, R, N, Replace A94 with Q, F, W, Y, P, or C; replace A95 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace A95 with D, E, H, Replace A96 with K, R, N, Q, F, W, Y, P, or C; replace G97 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A98; with D, E, H, K, R, A, G, I, L, S, T, Replace P99 with M, V, N, Q, F, W, Y, or C; replace P99 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Replace E100 with Y, P or C; replace L101 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q , F, W, Y, P, or C for T102; D, E, H, K, R, N, Q, F, W, Y, P, or C for A103; D, E, H, K , R, N, Q, F, W, Y, P, or C to replace G104; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace V105; use D , E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace K106; use D, E, H, K, R, N, Q , F, W, Y, P, or C for L107; D, E, H, K, R, N, Q, F, W, Y, P, or C for L108; D, E, H, K , R, N, Q, F, W, Y, P, or C to replace T109; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q , F, W, Y, or C for P110; D, E, H, K, R, N, Q, F, W, Y, P, or C for A111; D, E, H, K, R , N, Q, F, W, Y, P, or C to replace A112; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F , W, Y, or C to replace P113; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace R114; use D, Replace P115 with E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; with D, E, A, G, I, Replace H116 with L, S, T, M, V, N, Q, F, W, Y, P, or C; replace H116 with D, E, H, K, R, A, G, I, L, S, T, Replace N117 with M, V, F, W, Y, P, or C; replace S118 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, Replace S119 with H, K, R, N, Q, F, W, Y, P, or C; replace S119 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R120; D, E, H, K, R, N, Q, F, W, Y, P, or C for G121; D, E, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P, or C to replace H122; with D, E, A, G, I, L, S, T, M, V, N, Q , F, W, Y, P, or C for R123; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C Substitute N124; Substitute D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R125; Substitute D, E, A, G, I , L, S, T, M, V, N, Q, F, W, Y, P or C to replace R126; with D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A127; use D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F128; use D, E, H, Replace Q129 with K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; with D, E, H, K, R, N, Q, F, W , Y, P, or C to replace G130; to replace G130 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C P131; Substitution of E132 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Substitution of H, K, R, A, Replace E133 with G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace E133 with D, E, H, K, R, N, Q, F, W, Y , P, or C to replace T134; replace E135 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C to replace Q136; with H, K, R, A, G, I, L , S, T, M, V, N, Q, F, W, Y, P, or C for D137; D, E, H, K, R, N, Q, F, W, Y, P, or C Substitute V138; Substitute D139 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Substitute D, E, H, K , R, N, Q, F, W, Y, P, or C for L140; D, E, H, K, R, N, Q, F, W, Y, P, or C for S141; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace A142; with D, E, H, K, R, A, G, I, L, S, T, M , V, N, Q, F, W, Y, or C to replace P143; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F , W, Y, or C for P144; D, E, H, K, R, N, Q, F, W, Y, P, or C for A145; D, E, H, K, R, A , G, I, L, S, T, M, V, N, Q, F, W, Y, or C to replace P146; with D, E, H, K, R, A, G, I, L, S , T, M, V, N, Q, F, W, Y, or P, for C147; D, E, H, K, R, N, Q, F, W, Y, P, or C for L148 ; Replace P149 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Substitute D, E, H, Replace G150 with K, R, N, Q, F, W, Y, P, or C; replace G150 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, for C151; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R152 ; replace H153 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use D, E, H, K, R, Replace S154 with N, Q, F, W, Y, P, or C; replace S154 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, Replace Q155 with P or C; replace H156 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replace H156 with H, K, R , A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D157; with H, K, R, A, G, I, L, S, Replace D158 with T, M, V, N, Q, F, W, Y, P or C; replace D, E, H, K, R, A, G, I, L, S, T, M, V, F , W, Y, P, or C for N159; D, E, H, K, R, N, Q, F, W, Y, P, or C for G160; D, E, H, K, R , N, Q, F, W, Y, P or C to replace M161; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N162; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L163; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R164; D, E, H, K, R, A, G, I, L, S, T, M, V, F , W, Y, P, or C for N165; D, E, H, K, R, N, Q, F, W, Y, P, or C for I166; D, E, H, K, R , N, Q, F, W, Y, P, or C to replace I167; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y , P or C to replace Q168; replace D169 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use D, E , H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, replace C170; use D, E, H, K, R, Replace L171 with N, Q, F, W, Y, P, or C; replace L171 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, Replace Q172 with P or C; replace L173 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, N, Q, F , W, Y, P, or C for I174; D, E, H, K, R, N, Q, F, W, Y, P, or C for A175; H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D176; with D, E, H, K, R, N, Q, F, W, Y, Replace S177 with P, or C; replace D178 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace D, E , H, K, R, N, Q, F, W, Y, P, or C to replace T179; with D, E, H, K, R, A, G, I, L, S, T, M, V , N, Q, F, W, Y, or C for P180; D, E, H, K, R, N, Q, F, W, Y, P, or C for A181; D, E, H , K, R, N, Q, F, W, Y, P, or C to replace L182; H, K, R, A, G, I, L, S, T, M, V, N, Q, F , W, Y, P or C for E183; H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for E184; Replace K185 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; use H, K, R, A, G, I , L, S, T, M, V, N, Q, F, W, Y, P or C to replace E186; with D, E, H, K, R, A, G, I, L, S, T, Replace N187 with M, V, F, W, Y, P, or C; replace N187 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K188; D, E, H, K, R, N, Q, F, W, Y, P, or C for I189; D, E, H, K, R, N, Q, F, W, Y, P, or C for V190; D, E, H, K, R, N, Q, F, W, Y, P, or C for V191; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R192; D, E, H, K, R, A, G, I, L, S, T, M , V, F, W, Y, P or C to replace Q193; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace T194; use D, E, H, Replace G195 with K, R, N, Q, F, W, Y, P, or C; replace G195 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for Y196; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for F197; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F198; with D, E, H, K, R, N, Q, Replace I199 with F, W, Y, P, or C; replace Y200 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C ; replace S201 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E, H, K, R, A, G, I, L, S, Replace Q202 with T, M, V, F, W, Y, P or C; replace V203 with D, E, H, K, R, N, Q, F, W, Y, P, or C; use D, E , H, K, R, N, Q, F, W, Y, P, or C to replace L204; with D, E, H, K, R, N, Q, A, G, I, L, S, T , M, V, P, or C for Y205; D, E, H, K, R, N, Q, F, W, Y, P, or C for T206; H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace D207; with D, E, H, K, R, A, G, I, L, S, Replace P208 with T, M, V, N, Q, F, W, Y, or C; replace I209 with D, E, H, K, R, N, Q, F, W, Y, P, or C; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C to replace F210; use D, E, H, K, R, N, Replace A211 with Q, F, W, Y, P, or C; replace M212 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K , R, N, Q, F, W, Y, P, or C to replace G213; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y , P, or C for H214; D, E, H, K, R, N, Q, F, W, Y, P, or C for V215; D, E, H, K, R, N, Q , F, W, Y, P, or C to replace I216; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C Substitute Q217; Substitute D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R218; Substitute D, E, A, G, I , L, S, T, M, V, N, Q, F, W, Y, P, or C for K219; D, E, A, G, I, L, S, T, M, V, N , Q, F, W, Y, P, or C for K220; D, E, H, K, R, N, Q, F, W, Y, P, or C for V221; D, E, A , G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C to replace H222; use D, E, H, K, R, N, Q, F, W , Y, P, or C for V223; D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C for F224; D , E, H, K, R, N, Q, F, W, Y, P, or C to replace G225; replace G225 with H, K, R, A, G, I, L, S, T, M, V, N , Q, F, W, Y, P or C to replace D226; replace D226 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for E227; D, E, H, K, R, N, Q, F, W, Y, P, or C for L228; D, E, H, K, R, N, Q, F, W , Y, P, or C for S229; D, E, H, K, R, N, Q, F, W, Y, P, or C for L230; D, E, H, K, R, N , Q, F, W, Y, P, or C for V231; D, E, H, K, R, N, Q, F, W, Y, P, or C for T232; D, E, H , K, R, N, Q, F, W, Y, P, or C to replace L233; with D, E, H, K, R, N, Q, A, G, I, L, S, T, M , V, P, or C to replace F234; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace R235; use D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, replace C236; use D, E, H, K, R , N, Q, F, W, Y, P, or C to replace I237; with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y , P or C to replace Q238; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N239; use D, Replace M240 with E, H, K, R, N, Q, F, W, Y, P or C; replace M240 with D, E, H, K, R, A, G, I, L, S, T, M, V , N, Q, F, W, Y, or C to replace P241; with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K242; D, E, H, K, R, N, Q, F, W, Y, P, or C for T243; D, E, H, K, R, N, Q, F, W , Y, P, or C to replace L244; replace with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C P245; replace N246 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; use D, E, H, K , R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N247; use D, E, H, K, R, N, Q, F, W , Y, P, or C to replace S248; with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P, Replace C249; replace Y250 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace S251; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace A252; use D, E, H, K, R, N, Q, F, W, Y, P, or C for G253; D, E, H, K, R, N, Q, F, W, Y, P, or C Replace I254; replace A255 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for R256; D, E, H, K, R, N, Q, F, W, Y, P, or C for L257; H, K , R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace E258; with H, K, R, A, G, I, L, Replace E259 with S, T, M, V, N, Q, F, W, Y, P, or C; replace with D, E, H, K, R, N, Q, F, W, Y, P, or C G260; replace D261 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; use H, K, R, A, Replace E262 with G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace E262 with D, E, H, K, R, N, Q, F, W, Y , P, or C to replace I263; use D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C to replace Q264; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L265; with D, E, H, K, R, N, Q, F, W, Y, P, or C Replace A266; replace I267 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replace D, E, H, K, R, A, G, I, L, Replace P268 with S, T, M, V, N, Q, F, W, Y, or C; replace P268 with D, E, A, G, I, L, S, T, M, V, N, Q, F, Replace R269 with W, Y, P or C; replace E270 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N271; use D, E, H, K, R, Replace A272 with N, Q, F, W, Y, P, or C; replace A272 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C for Q273; D, E, H, K, R, N, Q, F, W, Y, P, or C for I274; D, E, H, K, R, N, Q, F , W, Y, P, or C to replace S275; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C to replace R276; D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C to replace N277; use D, E, H, K, R, Replace G278 with N, Q, F, W, Y, P, or C; replace G278 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, Replace D279 with P or C; replace D280 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replace D, E, Replace T281 with H, K, R, N, Q, F, W, Y, P, or C; replace T281 with D, E, H, K, R, N, Q, A, G, I, L, S, T, Replace F282 with M, V, P, or C; replace F283 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D, E, H, K, R, N, Q, F, W, Y, P, or C to replace G284; with D, E, H, K, R, N, Q, F, W, Y, P, Or C to replace A285; use D, E, H, K, R, N, Q, F, W, Y, P, or C to replace L286; use D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for K287; D, E, H, K, R, N, Q, F, W, Y, P, or C for L288; and /or replace L289 with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-α protein of the present invention can be routinely screened for Neutrokine-α and/or Neutrokine-αSV functional activity and/or physical properties (such as enhanced or reduced stability and/or solubility) as described herein and known in the art. ). Preferably, the resulting protein of the invention has enhanced and/or reduced Neutrokine-α and/or Neutrokine-αSV functional activity. More preferably, the resulting Neutrokine-α and/or Neutrokine-αSV protein of the present invention has more than one enhanced and/or decreased functional activity and/or physical property of Neutrokine-α and/or Neutrokine-αSV.

In another embodiment, the Neutrokine-α polypeptide of the present invention comprises more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids.

Amino acid substitutions can also alter the selective binding of ligands to cell surface receptors. For example, Ostade et al., Nature 361:266-268 (1993) describe mutations that cause TNF-a to bind selectively to only one of the two known TNF receptors. Since Neutrokine-α and Neutrokine-αSV are members of the TNF polypeptide family, mutations similar to those in TNF-α have similar effects in Neutrokine-α and/or Neutrokine-αSV.

Critical sites of ligand-receptor binding can also be determined by structural analysis, such as crystallography, NMR, or radioaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and Uos et al., Sci. 255:306-312 (1992)).

Since Neutrokine-α is a member of the TNF-related protein family, in order to regulate rather than completely eliminate the functional activity (such as biological activity) of Neutrokine-α, the sequence of amino acids in the conserved domain of TNF can be mutated. 1A and 1B (SEQ ID NO: 2) shown in the Gly191-Leu284 amino acid position, preferably all in this region, most or some TNF family members (such as TNF-α, TNF-β, LT-β, and Fas Ligand) are non-conserved amino acid residues (see Figure 2A-B). By making specific mutations in Neutrokine-α at positions where this conserved amino acid is typically found in related TNFs, the Neutrokine-α mutein will act as an antagonist and thus have the ability to inhibit lymphocyte (such as B cell) proliferation, differentiation and / or activation. Accordingly, polypeptides of the invention include Neutrokine-alpha mutants. This Neutrokine-α mutant comprises or consists of a fragment, variant or derivative of the full-length or preferred ectodomain of the Neutrokine-α amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2). The polynucleotides encoding the aforementioned Neutrokine-α mutants are also included in the present invention.

Since Neutrokine-αSV is a member of the TNF-related protein family, in order to regulate rather than completely eliminate the functional activity of Neutrokine-αSV, the sequence of amino acids in the TNF conserved domain can be mutated, and the amino acids are shown in Figures 5A and 5B (SEQ ID NO : 19) in the Gly172-Leu265 amino acid position, preferably in this region in all, most or some TNF family members (such as TNF-α, TNF-β, LT-β, and Fas ligand) are non-conserved amino acids residues (see Figure 2A-B). By making specific mutations in Neutrokine-αSV at positions where this amino acid is typically found in related TNFs, the Neutrokine-αSV mutein will act as an antagonist, thus having, for example, inhibition of lymphocyte (e.g. B cell) proliferation, differentiation and/or Activation. Accordingly, polypeptides of the invention include Neutrokine-αSV mutants. This Neutrokine-αSV mutant comprises or consists of a fragment, variant or derivative of the full-length or preferred ectodomain of the Neutrokine-αSV amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19). The polynucleotides encoding the aforementioned Neutrokine-αSV mutants are also included in the present invention.

In addition, those skilled in the art are aware that mutations directed to certain regions of the Neutrokine-α polypeptides of the present invention that are encompassed by regions not included in the Neutrokine-αSV polypeptide sequence may affect the observed functional activity (e.g., biological activity) of the Neutrokine-α polypeptides. The 19 amino acid residues found were inserted into the sequence (i.e. the Val142-Lys160 amino acid residues of the sequence shown in Figures 1A and 1B and SEQ ID NO: 2). More particularly, such residues of the Neutrokine-α polypeptide sequence that can be targeted for mutagenesis include, for example without limitation, the amino acid residues of the Neutrokine-α polypeptide sequence shown in SEQ ID NO: 2: V142; T143; Q144; D145; C146; L147; Q148; L149; I150; A151; D152; S153; E154; T155; P156; T157; I158; Q159; and K160.

Recombinant DNA methods known in the art (see eg DNA shuffling, supra) can be used to generate novel mutant proteins or muteins, including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or increased stability. In addition, they can be purified in high yields and exhibit better solubility than the corresponding natural polypeptides, at least under certain purification and storage conditions.

Accordingly, the present invention also covers derivatives and analogs of Neutrokine-alpha and/or Neutrokine-alphaSV which have one or more amino acid residues deleted, added or substituted to produce compounds more suitable for expression in the host cell of choice. , amplified Neutrokine-α and/or Neutrokine-αSV polypeptides. For example, cysteine residues can be deleted or substituted with other amino acid residues to eliminate disulfide bonds; N-linked glycosylation sites can be altered or eliminated, for example, to achieve expression of homologous products that are more readily Recovered and purified from yeast hosts known to have hyperglycosylation sites. Therefore, in Neutrokine-α and/or Neutrokine-αSV polypeptides, various amino acid substitutions at the first or third amino acid positions or both positions of one or more glycosylation recognition sequences, and/or at one or Amino acid deletions at the second amino acid position of multiple such recognition sequences will prevent Neutrokine-α and/or Neutrokine-αSV from glycosylation of the modified tripeptide sequence (see Miyajimo et al., EMBO Journal 5(6): 1193 -1197).

Additionally, one or more amino acid residues (eg, arginine and lysine residues) of the polypeptides of the invention may be deleted or substituted with other residues to eliminate undesired processing by proteases such as furin or Kexins. One possible consequence of this mutation is that the Neutrokine-alpha polypeptides of the invention are not cleaved and released from the cell surface.

In a specific embodiment, Lys132 and/or Arg133 of the Neutrokine-α sequence shown in SEQ ID NO: 2 are mutated to other amino acid residues or deleted together, to prevent or reduce the soluble form of Neutrokine-α from expressing Neutrokine-α Alpha cells are released. In a more specific embodiment, Lys132 of the Neutrokine-alpha sequence shown in SEQ ID NO: 2 is mutated to Ala132. In another embodiment, Arg133 of the Neutrokine-alpha sequence shown in SEQ ID NO: 2 is mutated to Ala133. These mutated proteins and/or polynucleotides encoding these proteins are useful, for example, in vitro therapy or gene therapy to engineer cells expressing Neutrokine-alpha polypeptides retained on the surface of engineered cells.

In a specific embodiment, Cys146 of the Neutrokine-alpha sequence shown in SEQ ID NO: 2 is mutated to other amino acid residues or deleted, for example, to help prevent or reduce Neutrokine-alpha polypeptide mutations when expressed in an expression system Oligomerization of the body (essentially as described in Example 1). In a specific embodiment, Cys146 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, Cys232 of the Neutrokine-alpha sequence shown in SEQ ID NO: 2 is mutated to other amino acid residues or deleted to help prevent or reduce Neutrokine-alpha polypeptide mutations when expressed in an expression system Oligomerization of the body (essentially as described in Example 1). In a specific embodiment, Cys232 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, Cys245 of the Neutrokine-alpha sequence shown in SEQ ID NO: 2 is mutated to other amino acid residues or deleted, for example to help prevent or reduce the Neutrokine-alpha polypeptide when expressed in an expression system Oligomerization of the mutants (essentially as described in Example 1). In a specific embodiment, Cys245 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the invention are preferably in isolated form, and preferably substantially purified. Recombinantly produced Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides can be substantially purified by the one-step procedure described by Smith and Johnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include the complete polypeptide encoded by the deposited cDNA (ATCC deposit number 97768), including the intracellular domain, the transmembrane domain and the extracellular domain of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA Polypeptide, the ectodomain of protein minus intracellular domain and transmembrane domain, the complete polypeptide of Fig. 1A and 1B (the 1-285 amino acid residues of SEQ ID NO: 2), the mature soluble polypeptide of Fig. 1A and 1B (SEQ ID NO: 134-285 amino acid residues of 2), the ectodomain of Figure 1A and 1B (73-285 amino acid residues of SEQ ID NO: 2) minus the intracellular domain and the transmembrane domain, and the above polypeptide Polypeptides having at least 80%, 85%, 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% similarity. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the present invention include the complete polypeptide encoded by the deposited cDNA (ATCC deposit number 203518), including the intracellular domain, the transmembrane domain and the extracellular domain of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA Polypeptide, the extracellular domain of the protein minus the intracellular domain and the transmembrane domain, the complete polypeptide of Figure 5A and 5B (1-266 amino acid residues of SEQ ID NO: 19), the extracellular domain of Figure 5A and 5B (SEQ ID NO : 73-266 amino acid residues of 2) minus the intracellular domain and the transmembrane domain, and at least 80%, 85%, 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% similarity polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other polypeptides of the present invention include at least 80% or 85%, preferably at least 90% or 95%, more Polypeptides which are at least 96%, 97%, 98% or 99% identical are preferred, and portions of such polypeptides having at least 30, preferably at least 50 amino acids are also included. Polynucleotides encoding such polypeptides are also encompassed by the present invention.

Other polypeptides of the present invention include at least 80% or 85%, preferably at least 90% or 95%, more Polypeptides which are at least 96%, 97%, 98% or 99% identical are preferred, and portions of such polypeptides having at least 30, preferably at least 50 amino acids are also included. Polynucleotides encoding such polypeptides are also encompassed by the present invention.

The "similarity percentage" between two polypeptides refers to the comparison with Bestfit program (Wisconsin sequence analysis software package, version 8 Genetics Computer Group for Unix, University Research Park, 575 Science Drive, Madison, WI53711) and setting default parameters The amino acid sequences of two polypeptides are used to determine the similarity range generated by the similarity. Bestit uses Smith and Waterman's local homologous sequence alignment (Advances in Applied Mathematics 2:482-498, 1981) to find the best segment of similarity between two sequences.

A polypeptide having an amino acid sequence that is, for example, at least 95% "identical" to the amino acid sequence of a reference Neutrokine-α and/or Neutrokine-αSV polypeptide means that the amino acid sequence of the polypeptide is identical to the reference sequence, except for the polypeptide sequence Up to 5 changes per 100 amino acids of the amino acid sequence of the reference Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide may be included. In other words, to obtain a polypeptide comprising an amino acid sequence that is at least 95% identical to the reference amino acid sequence, 5% of the amino acid residues in the reference sequence may be deleted or substituted with other amino acids, or 5% of the total number of amino acid residues in the reference sequence 5% of the amino acids could be inserted into the reference sequence. These changes in the reference sequence may occur at the amino- or carboxy-terminus of the reference sequence, or anywhere between these termini, interspersed between individual residues in the reference sequence or in one or more contiguous groups in the reference sequence.

In practice, whether any particular polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the following sequence can be determined using known computer programs such as the Bestfit program (as before) Determine that said sequence is, for example, the amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2), the amino acid sequence encoded by the preserved cDNA clone HNEDU15 (ATCC NO.97768), or their fragments, or for example Figure 5A and the amino acid sequence shown in 5B (SEQID NO: 19), the amino acid sequence or their fragments encoded by the preserved cDNA clone HDPMC52 (ATCCNO.203518). When using Bestfit or any other sequence comparison program to determine whether a particular sequence is, for example, at least 95% identical to a reference sequence of the invention, parameters are of course set so that the percent identity is calculated over the full-length reference sequence and allow for Homology gaps accounting for 5% of the total number of amino acid residues in the reference sequence.

In specific embodiments, the identity between the reference (reference) sequence (sequence of the invention) and the subject sequence, also called global sequence alignment, is determined using the FASTDB computer program (Comp. Determined by App.Biosei.6:237-245 (1990).The preferred parameters for FASTDB amino acid sequence comparison are: Matrix=PAM 0, K-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length =0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or target sequence length, get shorter.According to this embodiment, if the target sequence is due to N or The C-terminal deletion is not shorter than the reference sequence due to internal deletions, and the results are manually adjusted because the FASTDB program does not take into account the truncation of the N- and C-terminals of the target sequence when calculating the overall percent identity. For sequences that are truncated at the N- and C-termini of the target sequence, the percent identity is adjusted by calculating the number of residues in the reference sequence that do not match the corresponding target sequence at the N- and C-terminal ends of the target sequence, as the total number of residues in the reference sequence. The percentage of the number of bases. Determining whether a residue is matched is determined by the FASTDB sequence comparison result. Then this percentage is subtracted from the percentage identity calculated by the FASTDB program with specific parameters to obtain the final percentage range of identity. This The final percent identity can be used in the present invention. Only the N and C terminal residues of the target sequence are not matched with the reference sequence, and the percent identity needs to be manually adjusted. That is, only the residues of the reference sequence are at the farthest N of the target sequence and C-terminal residues. For example, a 90 amino acid residue target sequence is aligned with a 100-residue reference sequence to determine percent identity. The deletion occurs at the N-terminal Matching of the first 10 residues at the N-terminus. These 10 unpaired residues represent 10% of the sequence (number of unmatched residues at the N- and C-termini/total number of residues in the reference sequence), so this 10 % is subtracted from the percent identity calculated by the FASTDB program. If the remaining 90 residues are sufficiently matched, the final percent identity is 90%. In another embodiment, a 90-residue target sequence is matched with 100 Reference sequence alignment of residues. This deletion is an intrinsic deletion, so there are no residues at the N- and C-termini of the target sequence that are not matched to the reference sequence. In this case, the percent identity calculated by FASTDB is different from artificial Adjustment. Furthermore, only the residues located outside the N and C terminals of the target sequence do not match the reference sequence, as shown in the FASTDB sequence comparison, manual adjustment is required at this time. The present invention does not require other manual adjustments.

Uses of the polypeptides of the invention include, but are not limited to, as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known in the art. In addition, as described in detail below, the use of the polypeptide of the present invention includes, but is not limited to, the production of polyclonal and monoclonal antibodies for detecting the expression of Neutrokine-α and/or Neutrokine-αSV, or as an agent capable of enhancing or inhibiting Neutrokine-α and/or Or agonists and antagonists of Neutrokine-αSV function. The polypeptides of the present invention also have therapeutic effects as described below. Additionally, such polypeptides can be used in yeast two-hybrid systems to "capture" Neutrokine-alpha and/or Neutrokine-alphaSV binding proteins that are also candidate agonists and antagonists of the invention. The yeast two-hybrid system is described in Fields and Song, Nature 340:245-246 (1989). GMOs and "knockouts"

Polypeptides of the invention can also be expressed in transgenic animals. Animals of any species including, but not limited to, rats, mice, rabbits, hamsters, guinea pigs, pigs, piglets, goats, sheep, cows, and non-human primates such as baboons, monkeys, and chimpanzees can be used to generate transgenic animals. In a specific embodiment, the methods described herein and others known in the art are used to express a polypeptide of the invention in humans as part of gene therapy.

Any method known in the art can be used to introduce a transgene (ie, a polynucleotide of the invention) into an animal to generate a founder strain of the transgenic animal. Such methods include, but are not limited to, pronuclear microinjection (Paterson et al., Biotechnology Applied Microbiology 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology Technology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Patent No. 4873191 (1989)); Retrovirus-mediated gene transfer into the germ line (Vander putten et al., PNAS 82 : 6148-6152 (1985), in blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Molecular Cell Biology 3 : 1803-1814 (1983)); introduce the polynucleotide of the present invention with gene gun (see Ulmer et al., Science 259: 1745 (1993); introduce the nucleic acid construct into embryonic pluripotent stem cells and move the stem cells back in blastocysts; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); et al. For a review of these methods, see Gordon, "Transgenic Animals", Int'l Rev Cytol. 115:171- 229 (1989), which is incorporated by reference in its entirety. Also see U.S. Patent No. 5,464,764 (Capecchi et al., Positive/negative selection methods and vectors); U.S. Patent No. 5,631,153 (Capecchi et al., Cells containing predetermined genome modifications and Non-human organisms and positive/negative selection methods and vectors for their production); U.S. Patent No. 4,736,866 (Leder et al., Transgenic non-human animals); and U.S. Patent No. 4,873,191 (Wagner et al., Genetic transformation of fertilized eggs); the above documents Both are incorporated by reference in their entirety.

Any method known in the art can be used to produce transgenic clones containing polynucleotides of the invention, e.g., transfer of nuclei to cultured embryonic, fetal or adult cells induced to a quiescent state (Campell et al. , Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The invention provides transgenic animals that carry the transgene in all of their cells, as well as animals that carry the transgene in some but not all of their cells, ie, mosaic or chimeric animals. This transgene can be integrated as a single transgene or in multiple copies such as concatemers, for example in head-to-head tandem or head-to-tail tandem. Transgenes can also be selectively introduced into and activated in specific cell types, such as taught by Lasko et al. (Lasko et al., Proc. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such cell type specific activation are dependent on the respective particular cell type and will be apparent to those skilled in the art. Gene targeting is preferred when integration of the polynucleotide transgene into the chromosomal location of an endogenous gene is desired. In short, when this method is used, a vector containing some nucleotide sequence homologous to an endogenous gene is designed, integrated into the nucleotide sequence of the endogenous gene and its function is disrupted by using Homologous recombination of chromosomal sequences. Transgenes can also be selectively introduced into specific cell types, thus inactivating endogenous genes only in that cell type, eg as taught by Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such cell type specific inactivation will depend on the respective particular cell type and will be apparent to those skilled in the art. In addition to expressing the polypeptides of the invention in a ubiquitous or tissue-specific manner in transgenic animals, it will be within the purview of those skilled in the art to create constructs that modulate expression of the polypeptides by various other means (e.g. developmentally or chemically regulated expression) It's also a breeze.

Once transgenic animals have been produced, expression of the recombinant gene can be analyzed using standard methods. Animal tissues can be initially screened by Southern blot analysis or PCR to verify whether transgene integration has occurred. Transgene mRNA expression levels in tissues of transgenic animals can also be determined by methods including, but not limited to, Northern blot analysis, in vivo hybridization analysis, reverse transcriptase-PCR (rt-PCR) on tissue samples obtained from the animal; and TagMan PCR. Tissue samples expressing the transgene can also be evaluated immunocytochemically or immunohistochemically with antibodies specific for the transgene product.

Once founder animals are produced, they can be bred, inbred, outbred, or cross-bred to produce a particular population of animals. Examples of such breeding methods include, but are not limited to, outbreeding founder animals with more than one integration site to create segregated lines; inbreeding segregated lines to produce transgenic compounds that express the transgene at high levels because each Due to the cumulative effect of transgene expression; cross-breeding heterozygous transgenic animals to generate animals homozygous for a given integration site to increase expression and eliminate the need to screen animals by DNA analysis; cross-breed segregating homozygous lines to generate heterozygotes or homozygous strains of the compound; propagating the transgene in a different background suitable for the test model; and propagating the transgenic animal into other animals carrying a different transgene or knockout mutation.

The uses of the transgenic and "knockout" animals of the present invention include, but are not limited to, elaborating the biological functions of Neutrokine-α and/or Neutrokine-αSV polypeptides, researching diseases related to abnormal expression of Neutrokine-α and/or Neutrokine-αSV, and Screening for effective compounds that ameliorate this disease serves as an animal model system.

In additional embodiments of the invention, genetically engineered cells expressing or not expressing (eg, knocking out) a polypeptide of the invention are administered to a patient in vivo. Such cells may be obtained from a patient (i.e., animal, including human) or MHC-compatible donor, and may include, but are not limited to, fibroblasts, bone marrow cells, blood cells (such as lymphocytes), adipocytes, muscle cells, endothelial cells wait. The cells are genetically engineered in vitro by recombinant DNA methods, and the coding sequence of the polypeptide of the present invention is introduced into the cell, or the coding sequence of the polypeptide of the present invention and/or the endogenous regulatory sequences related thereto are destroyed, such as by transduction (using Viral vectors, preferably vectors that integrate the transgene into the cell genome), or transfection methods include but are not limited to using plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. A coding sequence for a polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or a promoter/enhancer for the expression, preferably secretion, of the polypeptide of the invention. Engineered cells that express, and preferably secrete, a polypeptide of the invention can be introduced systemically into a patient, such as in the circulation or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, for example, genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft Implantation (see, eg, Anderson et al., US Patent No. 5,399,349; and Mulligan and Wilson, US Patent No. 5,460,959, both incorporated by reference).

When the cells to be administered are non-self or non-MHC-compatible cells, they can be administered by a method using a well-known tissue host to generate an immune response to the introduced cells. For example, cells can be introduced in capsule form, allowing immediate exchange of components with the extracellular environment without allowing the introduced cells to be recognized by the host immune system.

Antibody

Other polypeptides of the present invention relate to antibodies and T cell antigen receptors (TCR), which immunospecifically bind to the polypeptides, polypeptide fragments or variants of SEQ ID No: 2 and/or SEQ ID No: 19 of the present invention, and/ or epitopes (determined by immunoassays that analyze specific antibody-antigen binding) well known in the art. Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, Fab expression libraries Fragments generated, anti-idiotypic (anti-Id) antibodies (including anti-idiotypic antibodies raised against antibodies of the invention) and epitope-binding fragments of any of the above antibodies. The term "antibody" refers to immunoglobulin molecules and immunologically active portions thereof, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (such as IgG, IgE, IgM, IgD, IgA, and IgY), class (such as IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule. Immunoglobulins can be of heavy or light chain. A collection of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains can be paired with either kappa or lambda forms of light chains.

Most preferred antibodies are antibody fragments of the invention that bind human antigens, including but not limited to Fab, Fab', and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising VL or VH domains. Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable region alone or in combination with all or part of the following regions: hinge region, CH1, CH2, and CH3 domains. The invention also includes antigen-binding fragments that also comprise any combination of variable and hinge, CH1, CH2, and CH3 domains. Antibodies of the invention may be from any animal including birds and mammals. Preferably, the antibody is human, murine (eg rat and mouse), donkey, rabbit, goat, guinea pig, camel, horse or chicken. "Human" antibodies as used herein include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or from transgenes using one or more human immunoglobulins and which do not express endogenous immunoglobulins. Antibodies from animals are as previously described and described in Kucherlapati et al., US Patent No. 5,939,598.

Antibodies of the invention may be monospecific, bispecific, triple specific or more specific. Multispecific antibodies may be specific for different epitopes of a polypeptide of the invention, or may be specific for a polypeptide of the invention as well as a heterologous epitope, such as a heterologous polypeptide or a solid support. See, for example, PCT Publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunology 147:60-69 (1991); U.S. Patent Nos. 4,474,893; Kostelng et al., J. Immunol. 148: 1547-1553 (1992).

Antibodies of the present invention may be described in terms of the epitope or part of the polypeptide of the present invention that they recognize or specifically bind to. The epitope or part of the polypeptide can be illustrated, for example, by N-terminal and C-terminal position, size of contiguous amino acid residues, or listed in tables and figures. Antibodies that specifically bind any epitope or polypeptide of the invention may also be excluded. Thus, the invention includes antibodies that specifically bind a polypeptide of the invention, and allows the exclusion of identical antibodies.

In specific embodiments, an antibody of the invention binds to a polypeptide comprising the amino acid sequence of SEQ ID No: 2: Phe115-Leu147, Ile150-Tyr163, Ser171-Phe194, Glu223-Tyr246, and Ser271-Phe278. In another specific embodiment, the antibody of the present invention binds to a polypeptide comprising the following amino acid sequence: amino acids of Phe115-Leu147, Ile150-Tyr163, Ser171-Phe194, Glu223-Tyr246, and Ser271-Phe278 of SEQ ID No: 2 sequence. In a preferred embodiment, the antibody of the present invention binds to a polypeptide comprising Glu223-Tyr246 of SEQ ID No:2. In another preferred embodiment, the antibody of the present invention binds to a polypeptide consisting of Glu223-Tyr246 of SEQ ID No:2. In a more preferred embodiment, the antibody of the present invention binds to a polypeptide consisting of Phe230-Asn242 of SEQ ID No:2. In another preferred embodiment, the antibodies of the present invention inhibit one or more biological activities of Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention by specific binding. In a more preferred embodiment, the antibodies of the invention inhibit Neutrokine-α and/or Neutrokine-αSV mediated B cell proliferation.

Antibodies of the invention may also be described or illustrated in terms of their cross-reactivity. The invention includes antibodies that do not bind any other analogs, orthologs, or homologues of the polypeptides of the invention. The present invention also includes combinations having at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% identity (using methods known in the art and Antibodies to polypeptides calculated by methods described herein). In specific embodiments, the antibodies of the invention cross-react with rat, mouse and/or rabbit homologues of human proteins and their corresponding epitopes. The present invention also includes not binding to the identity of the polypeptide of the present invention is less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% (use known in the art Methods and antibodies to polypeptides calculated by the methods described herein). In a specific embodiment, the aforementioned cross-reactivity is with respect to any one specific antigenic or immunogenic polypeptide, or a combination of 2, 3, 4, 5 or more specific antigenic and/or immunogenic polypeptides . The invention also includes antibodies that bind a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide of the invention under hybridization conditions (as described herein). Antibodies of the invention can also be described or described in terms of their binding affinity for polypeptides of the invention. Preferred binding affinities include those with dissociation constants or Kd less than 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 M, 10 ^-6 M, 5 x 10 ^-7 M, 10 ^-7 M, 5× ^10-8 M, ^10-8 M, 5× ^10-9 M, ^10-9 M, 5× ^10-10 M, ^10-10 M, 5× ^10-11 M, ^10-11 M, 5 x 10 ^-12 M, 10 ^-12 M, 5 x ^{10 -13 M} , 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M, or 10 ^-15 M antibodies .

The invention also provides antibodies that competitively inhibit binding of the antibody to an epitope of the invention, as determined by any method known in the art to determine competitive binding, eg, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50%.

The antibodies of the invention can act as agonists or antagonists of the polypeptides of the invention. For example, the invention includes antibodies that partially or fully disrupt the interaction of a receptor/ligand with a polypeptide of the invention. Preferably, an antibody of the invention binds an antigenic epitope described herein, or a portion thereof. The invention includes receptor-specific antibodies and ligand-specific antibodies. Also included in the invention are receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (ie, signaling) can be determined by other methods described herein or known in the art. For example, receptor activation can be determined by detecting phosphorylation of the receptor or its substrates (eg, tyrosine or serine/threonine) by immunoprecipitation followed by Western blot analysis (as described above). In specific embodiments, there is provided an antibody that inhibits ligand activity or receptor activity by at least 95%, 90%, 85%, 80%, 75%, 70% of the activity in the absence of the antibody, 60%, or 50%.

Also included in the invention are receptor-specific antibodies that prevent ligand binding and receptor activation, as well as antibodies that recognize the receptor-ligand complex, and preferably do not specifically recognize unbound receptor or unbound ligand. Also included in the invention are neutralizing antibodies that bind a ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind the ligand, thereby preventing activation of the receptor but do not prevent binding of the ligand to the receptor. The invention additionally includes antibodies that activate receptors. These antibodies may act as receptor agonists, ie enhance or activate the biological activity of all or part of ligand-mediated receptor activation, for example by inducing receptor dimerization. Antibodies can act as agonists, antagonists or inverse agonists to demonstrate their biological activity, including the specific biological activity of the peptides of the invention. The above antibody agonists can be produced by methods known in the art. See, eg, PCT Publication WO 92/40281; US Patent No. 581109; Deng et al., Blood 92(6):1981-1988 (1998); Cher et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., Journal of Immunology 161(4): 1786-1794 (1998); Zhu et al., Cancer Research 58(15): 3209-3214 (1998); Yoon et al., Journal of Immunology 160(7): 3170-3179 (1998); Prat et al., J. Cell Sci. 111 (Pt2): 237-247 (1998); Pitard et al., J. Immunol. Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4): 233-241 (1997); Carlson et al., J. Biol. Chem. 272(17): 11295-11301 (1997); Taryman et al., Neuron 14(4): 755-762 (1995); Muller et al., Structure 6(9): 1153- 1167 (1998); Bartunek et al., Cytokine 8(1): 14-20 (1996) (all incorporated by reference in their entirety).

Antibodies of the invention can be used, for example, but not limited to, to purify, detect and target polypeptides of the invention, including in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies can be used in immunoassays to quantitatively and qualitatively determine the level of a polypeptide of the invention in a biological sample. See, eg, Harlow et al., Antibody Laboratory Manual (Published by Cold Spring Harbor Laboratory, Vol. II, 1988) (incorporated by reference in its entirety).

As described in more detail below, the antibodies of the invention may be used alone or in combination with other compositions. Antibodies can be fused at the N- or C-terminus to heterologous polypeptides, or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the invention can be recombinantly fused or conjugated to molecules used as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides or toxins. See, eg, PCT Publication WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.

Antibodies of the invention include modified derivatives, ie, covalently attached to the antibody by any type of molecule such that the covalent attachment does not prevent the antibody from producing an anti-idiotypic response. Antibody derivatives include, for example but without limitation, antibodies modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and Cell ligands or other protein linkages, etc. Any chemical modification can be performed by known methods, including but not limited to special chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives may contain one or more non-canonical amino acids.

Antibodies of the invention can be produced by any method known in the art. Polyclonal antibodies to corresponding antigens can be produced by various methods well known in the art. For example, the invention can be uniquely administered to various host animals including, but not limited to, rabbits, rats, mice, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to enhance the rabbit immune response depending on the host species, adjuvants include but are not limited to Freund's (complete or incomplete) adjuvants, inorganic gels such as alumina, surfactants such as lysolecithin, and more Alcohols, polyanions, peptides, oil latex, keyhole limpet hemocyanin, dinitrophenol, and potentially effective human adjuvants such as BCG (BCG), and Corynebacterium pumilus. These adjuvants are well known in the art.

Monoclonal antibodies can be prepared by various methods known in the art, including the use of hybridoma, recombinant, and phage display methods, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma methods, including those known in the art and such as Harlow et al., Antibody Laboratory Manual (Cold Spring Harbor Laboratory Press, Vol. 2, 1988); Hammerling et al., Monoclonal Antibodies and T cell hybridoma 563-681 (Elsevier, N.Y. 1981) (incorporated by reference in its entirety). The term "monoclonal antibody" is not limited to antibodies produced by the hybridoma method. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and does not refer to the method by which it is produced.

A "monoclonal antibody" may comprise or consist of two proteins, a heavy chain and a light chain.

The methods for producing and screening specific antibodies by hybridoma methods are well known in the art, and are described in the examples (eg Example 9). In a non-limiting example, cells unique to the invention or expressing such unique can be used for seeding. Once an immune response is detected, eg, antibodies specific for the antigen are detected in the mouse sera, the mouse is dissected and splenocytes are isolated. The spleen cells are then fused to any suitable myeloma cells, such as cells of the SP20 cell line from ATCC, by well known methods. Hybridomas were selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding the unique antibodies of the invention. Ascites fluid, which usually contains high levels of antibodies, can be produced by inoculating mice with positive hybridoma clones.

Accordingly, the present invention provides methods for producing monoclonal antibodies and antibodies produced by such methods, the methods comprising culturing hybridoma cells secreting antibodies of the present invention, wherein preferably the hybridomas are produced by fusion of spleen cells and myeloma cells Yes, splenocytes were isolated from mice immunized with an antigen of the invention, and the hybridomas obtained from the fusion were screened for hybridoma clones secreting antibodies unique to the invention.

Antibody fragments that recognize specific epitopes can be generated by known methods. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic digestion of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments), or trypsin (to produce F(ab')2 fragments). ). The F(ab')2 fragment contains the variable region, the constant region of the light chain and the CH1 domain of the heavy chain.

For example, antibodies of the invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In specific embodiments, such phage can be used to display antigen binding domains expressed from repertoire or combinatorial antibody libraries. Phage expressing an antigen-binding domain that binds the corresponding antigen can be selected or identified using antigen, such as labeled antigen or antigen bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages comprising fd and M13 binding domains expressed from phages with Fab, Fv or disulfide bond stabilized Fv antibodies recombinantly fused to phage gene III or gene VIII proteins domain. Phage display methods that can be used to produce antibodies of the invention include, for example, those methods described in the following references: e.g., Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. ); Kettleborough et al., European Journal of Immunology 24:952-958 (1994); Persic et al., Genes 187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/20401; , 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743, and 5969108, all of which are incorporated by reference in their entirety.

As described in the above references, following phage selection, the antibody coding regions in the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, as detailed below. For example, methods for recombinantly producing Fab, Fab' and F(ab') fragments can also be applied by methods known in the art, such as PCT Publication WO 92/22324; Mullinax et al., Biotechnology 12(6):864-869 ( 1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988), all of which are incorporated by reference in their entirety.

Methods that can be used to generate single-chain Fvs and antibodies include, for example, U.S. Patents 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Sbu et al., PNAS 90:7995-7999 (1993); and SKerra et al., Sci. 240:1038-1040 (1988) as those described. For some applications, including the use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies is preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from animals of different species, eg, an antibody having variable regions derived from a murine monoclonal antibody and human immunoglobulin constant regions. Methods for generating chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., Biotechnology 4:214 (1986); Gillies et al. (1989), J. Immunol. Methods 125:191-202; described in U.S. Pat. . Humanized antibodies are antibody molecules derived from antibodies of a non-human species that bind a desired antigen having one or more complementarity determining regions (CDRs) of the non-human species and the framework regions of a human immunoglobulin molecule. Typically, framework residues within the human framework regions will be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling the interactions of CDRs with framework residues to identify framework residues important for antigen binding and performing sequence alignments to identify unique framework residues at particular positions (See, eg, Queen et al., US Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), incorporated by reference in their entirety). Antibodies can be humanized by a variety of methods known in the art, including, for example, CDR shifting (EP239400; PCT Publication; /09967 US Patent Nos. 5225539, 5530101, and 5585089), Veneering or resurfacerization (EP592106; EP 519596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); Roguska et al., PNAS 91:969-973(1994)), and chain Reorganization (US Patent No. 5565332).

Fully human antibodies are particularly desirable for treating patients. Human antibodies can be produced by various methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887, and 4,716,111; and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, Each document is incorporated by reference in its entirety.

Human antibodies can also be produced in transgenic mice that do not express functional endogenous immunoglobulins, but instead express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be randomly introduced or homologously recombined into murine embryonic stem cells. Alternatively, in addition to the human heavy and light chain genes, human variable, constant and multivariable regions can be introduced into mouse embryonic stem cells. The murine heavy and light chain immunoglobulin genes can be non-functionally separated or simultaneously introduced into the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal manner with a selected antigen, such as all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from transgenic mice immunized by conventional hybridoma methods. Human immunoglobulin transgenes are rearranged during B-cell differentiation by transgenic mice, followed by class switching and somatic mutation. Thus, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies using this method. A general study of this method of producing human antibodies is described in Lonberg and Huszar, Immunol. Res. Int. 13:65-93 (1995). Methods for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies are detailed in, for example, PCT Publications WO 98/24893, WO 92/01047, WO 96/34096, WO 96/33735; European Patent No. 0598877; U.S. Patent Nos. 5413923, 5625126, 5633425, 5569825, 5661016, 5545806, 5814318, 5885793, 5916771 and 5939598, all of which are incorporated by reference in their entirety. In addition, Abgenix Corporation (Freemont, CA) and Genpharm Corporation (San Jose, CA) provide human antibodies directed against selected antigens, produced using methods similar to those described above.

Fully human antibodies that recognize selected epitopes can be generated by a method known as "directed selection". In this approach, selected non-human monoclonal antibodies, such as murine antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Biotechnology 12:899-903 (1988)).

In addition, antibodies to polypeptides of the invention can be used to generate anti-idiotypic antibodies that "mimic" polypeptides of the invention, using methods well known in the art (see, e.g., Greenspan and Bona, J. FASEB 7(5):437-444, (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies that bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate "mimetic" polypeptide multimerization and/or binding domains, and thus bind and neutralize Anti-idiotypes of polypeptides and/or their ligands. Such neutralizing anti-idiotypic antibodies or Fab fragments of such anti-idiotypic antibodies can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or bind its ligand/receptor, thereby blocking its biological activity.

Polynucleotides Encoding Antibodies

The present invention also provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or low stringency hybridization conditions (as described above) to a polynucleotide encoding an antibody that preferably specifically binds to a polypeptide of the invention, more preferably has The polypeptide of the amino acid sequence shown in SEQ ID NO:2. In another preferred embodiment, the antibody specifically binds a polypeptide having the amino acid sequence of SEQ ID NO: 19. In another preferred embodiment, the antibody specifically binds a polypeptide having the amino acid sequence of SEQ ID NO: 23. In another preferred embodiment, the antibody specifically binds a polypeptide having the amino acid sequence of SEQ ID NO: 28. In another preferred embodiment, the antibody specifically binds a polypeptide having the amino acid sequence of SEQ ID NO:30.

A polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of a polynucleotide can be determined by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (as described by Kutmeier et al., Biotechnology 17:242 (1994)), briefly, This involves synthesizing overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, polynucleotides encoding antibodies may be generated from nucleic acids from suitable sources. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the immunoglobulin-encoding nucleic acid can be synthesized chemically, or using a gene sequence that is compatible with the particular gene sequence to be identified, e.g., from a cDNA library encoding the antibody. Synthetic primers hybridizing to the 3' and 5' ends of cDNA clones were obtained from appropriate sources by PCR amplification or by cloning with oligonucleotide probes specific for the sequence (e.g. antibody cDNA libraries, or generated from expression Any tissue or cell for the antibody such as a cDNA library of hybridoma cells selected for expressing the antibody of the invention, or nucleic acid, preferably polyA+RNA, isolated from any tissue or cell expressing the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector by any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody are determined, the nucleotide sequence of the antibody can be manipulated by methods well known in the art, such as recombinant DNA methods, site-directed mutagenesis, PCR, etc. (see, for example, Sambrook et al., 1990, Molecular Cloning Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., 1998, General Methods in Molecular Biology, John Wiley and Sons, NY, said documents are incorporated by reference in their entirety), to generate Antibodies, for example by making amino acid substitutions, deletions and/or insertions.

In a specific embodiment, the amino acid sequences of the heavy and/or light chain variable domains are examined to identify the sequences of the complementarity determining regions (CDRs), by methods well known in the art, such as comparison to other heavy and The known amino acid sequence of the light chain variable region to identify regions of high sequence variability. Using conventional recombinant DNA methods, one or more CDRs can be inserted into a framework region, such as into a human framework region, to humanize a non-human antibody, as previously described. The framework regions may be naturally occurring or consensus framework regions, and are preferably human framework regions (see, eg, Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a list of human framework regions). Preferably, the polynucleotide produced by combining the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, one or more amino acid substitutions may be made within the framework regions, as previously described, and preferably, such amino acid substitutions improve binding of the antibody to its antigen. Additionally, this method can be used to generate amino acid substitutions or deletions of one or more variable region cysteine residues that participate in intrachain disulfide bonds to generate antibody molecules lacking one or more intrachain disulfide bonds. Other variations of polynucleotides are also encompassed by the invention.

Alternatively, methods of producing "chimeric antibodies" can be used (Morrison et al., Proc. 1985)), by splicing the gene from a murine antibody molecule of appropriate antigen specificity with the gene from a human antibody molecule of appropriate biological activity. As mentioned previously, a chimeric antibody is a molecule in which different parts are derived from animals of different species, such as those having variable regions derived from murine mAbs and human immunoglobulin constant regions, eg humanized antibodies.

Alternatively, methods for producing single-chain antibodies can be used (U.S. Patent No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. 334:544-54 (1989), produce single-chain antibody.Single-chain antibody is to connect the heavy chain and light chain fragment of Fv region by amino acid bridge, produces single-chain antibody.It can also be used to assemble functional Fv fragment in Escherichia coli Methods (Skerra et al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any synthetic antibody known in the art, especially by chemical synthesis or preferably by recombinant expression methods. The recombinant expression of the antibody of the present invention or its fragment, derivative or analog (such as the heavy chain or light chain of the antibody of the present invention, or the single chain antibody of the present invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody . Once a polynucleotide encoding an antibody molecule of the invention or its heavy or light chain or a portion thereof (preferably a portion containing a heavy or light chain variable domain) has been obtained, the vector for producing the antibody molecule can be produced by methods well known in the art. Produced by recombinant DNA methods. Thus, described herein are methods of making proteins by expressing polynucleotides comprising antibody coding sequences. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed by methods well known in the art. These methods include, for example, in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. The present invention thus provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention or its heavy or light chain, or heavy or light chain variable region, operably linked to a promoter. Such vectors may include nucleotide sequences encoding constant regions of antibody molecules (see, e.g., PCT Publication WO86/05807; PCT Publication WO89/01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibody may be cloned into This vector allows expression of the entire heavy or light chain.

The expression vector is transferred into host cells by a conventional method, and the transfected cells are cultured by a conventional method to produce the antibody of the present invention. Thus, the invention includes host cells comprising a polynucleotide encoding an antibody of the invention, its heavy or light chain, or a single chain antibody of the invention operably linked to a heterologous promoter. In preferred embodiments for the expression of diabodies, vectors encoding the heavy and light chains can be co-expressed in host cells to express the entire immunoglobulin molecule, as described below.

A variety of host expression vector systems can be used to express the antibody molecules of the invention. Such a host expression system represents a vector by which the corresponding coding sequence can be produced and subsequently purified, but also represents a cell which, when transformed or transfected with the appropriate nucleotide coding sequence, expresses the present gene in situ. Invented antibody molecule. These host expression systems include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA containing antibody coding sequences, plasmid DNA or cosmid DNA expression vectors (such as Escherichia coli, Bacillus subtilis); Yeast transformed with expression vectors (such as Saccharomyces, Pichia pastoris); insect cell systems infected with recombinant virus expression vectors (such as baculovirus) containing antibody coding sequences; infected with recombinant virus expression vectors containing antibody coding sequences (such as Cauliflower mosaic virus, CaMV; Tobacco mosaic virus TMV) infected, or plant cell systems transformed with recombinant plasmid expression vectors (such as Ti plasmids) containing antibody coding sequences; or with promoters derived from mammalian cell genomes (e.g. metallothionein promoter) or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3 cells). Preferably, bacterial cells such as Escherichia coli, more preferably, especially eukaryotic cells expressing the entire recombinant antibody molecule are used for expression of the recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), are effective expression systems for antibodies in combination with vectors such as the major mid-early gene promoter factors of human cytomegalovirus (Foecking et al., Gene 45:101 (1986) ; Cockett et al. Bio/Technology 8:2 (1990).

In bacterial systems, a number of advantageous expression vectors can be chosen depending on the antibody molecule to be expressed. For example, when large quantities of such proteins are produced, vectors for direct high-level expression of fusion protein products that are readily purified are required for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the Escherichia coli expression vector pUR278 (Ruther et al., EMBO Journal 2: 1791 (1983)), wherein the antibody coding sequence is individually linked in the vector with the LacZ coding region in frame to produce a fusion protein; the pIN vector ( Inouye and InouYe, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)) et al. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). Typically, this protein is soluble and easily purified from lysed cells by adsorption and binding to the matrix glutathione-agarose beads followed by elution in the presence of free glutathione . pGEX vectors include thrombin or Factor Xo proteolysis sites so that the cloned target gene product can be released from the GST component.

In insect systems, Autographa californica polynucleated polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned separately into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems are available. When adenovirus is used as the expression vector, the corresponding antibody coding curve can be linked to the adenovirus transcription/translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion in non-essential regions of the viral genome (such as the El or E3 regions) produces recombinant viruses that are viable and capable of expressing antibody molecules in infected hosts (see, for example, Logan and Shenk, Proc. -359 (1984)). Specific initiation signals may also be required for efficient translation of antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in frame with the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, such as natural and synthetic. Expression efficiency can be enhanced by inclusion of appropriate transcriptional enhancer factors, transcriptional terminators, etc. (see Bitther et al., Methods in Enzymology 153:51-544 (1987)).

In addition, host cell strains can be selected that regulate the expression of the inserted sequences or that modify and process the gene product in a specifically desired manner. Such modification (eg, glycosylation) and processing (eg, cleavage) of protein products are important for protein function. Different host cells have specialized and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the foreign protein to be expressed. For this purpose, eukaryotic host cells with the correct cellular machinery for processing the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, especially breast cancer cell lines and BT 483, Hs 578T, HTB2, HT20, and T47D, and normal mammary gland Cell lines such as CRL 7030 and Hs 578Bst.

For long-term high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express antibody molecules can be genetically engineered. Unless an expression vector containing a viral origin of replication is used, host cells can be transformed with DNA controlled by appropriate expression control elements (such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers . After the introduction of exogenous DNA, the engineered cells are grown in nourishing media for 1-2 days and then switched to selective media. A selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromatin and grow to form colonies that can then be cloned and expanded into cell lines. This method can be used to genetically engineer cell lines expressing antibody molecules. Such engineered cell lines are particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

Many selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. )), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980) genes can be used in tk-, hgprt- or aprt- cells, respectively. Likewise, antimetabolite resistance can be used as a basis for selection of : dhfr, which confers resistance to methotrexate (Wigler et al, PNAS 77:357 (1980); O'Hare et al, PNAS 78:1527 (1981)); gpt, which confers mycophenolic acid resistance ( Mulligan & Berg, PNAS 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G418 (Clinical Pharmacology 12:488-505; Wu and Wu, Biotherapeutics 3:87-95 (1991); Tolstoshev , Analytical Research in Drug Toxicity 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Analytical Research in Biochemistry 62: 191-217 (1993); May, 1993, TIB, TECH 11(5):155-215); and hygro, which confers hygromycin resistance (Santerre et al., Gene 30:147 (1984). Recombinant DNA methods known in the present invention can be routinely used to select for the desired recombinant Cloning, such as by Ausubel et al., (ed.), General Methods in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, A Laboratory Manual of Gene Transfer and Expression, Stockton Press, NY (1990); and Dracopoli et al. ( eds), General Methods in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colbene-Garapin et al., J. Mol. Biol. 150:1 (1981), all of which are hereby incorporated by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (see Bebbington, and Hentschel, Expression of Cloned Genes in Mammalian Cells Using Vectors Based on Gene Amplification, DNA Cloning, Vol. III (Science Press, New York, 1987 )). When the marker in the vector system expressing the antibody is amplifiable, increasing the level of the inhibitor in the host cell culture will increase the copy number of the marker gene. Antibody production is also increased because the amplified region is associated with the antibody gene (Crouse et al., Mol. Cell Biol. 3:257 (1983)).

Host cells can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker, which enables identical expression of the heavy and light chain polypeptides. Alternatively, a vector encoding and capable of expressing heavy and light chain polypeptides may be used. In this case, the light chain should be placed before the heavy chain to avoid excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. The coding sequence for the light chain may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by animal, chemical synthesis, or recombinant expression, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, Affinity chromatography, especially by specific antigen affinity chromatography followed by protein A, and size column chromatography), centrifugation, differential solubility, or by any other standard method of protein purification. In addition, antibodies or fragments thereof of the invention may be fused to other heterologous sequences described herein or known in the art to facilitate purification.

The invention encompasses recombinant fusion or chemical conjugation (including covalent and non-covalent transformation) of the polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid polypeptides) to produce fusion proteins. Fusion need not be direct, but can also occur through linker sequences. Antibodies may be specific for an antigen other than a polypeptide of the invention (or a portion thereof, preferably a polypeptide of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids). For example, antibodies can be used to target a polypeptide of the invention to a particular type of cell in vitro or in vivo by fusing or conjugating the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Antibodies fused or conjugated to polypeptides of the invention are also useful in in vitro immunoassays and purification methods. See, eg, Harbor et al., supra, and PCT Publication WO93/21232; EP 439095; Naramura et al., Immunology Communications, 39:91-99 (1994); U.S. Patent 5474981; Gillies et al., PNAS 89:1428-1432 (1992) ; Fecl et al., J. Immunol. 146: 2446-2452 (1991); all incorporated herein by reference in their entirety.

The invention also includes a composition comprising a polypeptide of the invention fused or conjugated to an antibody domain other than a variable region. For example, a polypeptide of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The portion of an antibody fused to a polypeptide of the invention may include a constant region, a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain, or any combination of all domains or a portion thereof. Polypeptides may also be fused or conjugated to portions of the antibodies described above to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through disulfide bonds between the Fc portions. Higher multimeric forms can be produced by fusing the polypeptide to parts of IgA and IgM. Methods for fusing or conjugating polypeptides of the invention to antibodies are known in the art. 5622929; 5359046; 5349053; 5447851; 5112946; EP 307434; EP367166; PCT publications WO 96/04388; ); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl Academia Sci. 89:11337-11341 (1992), all of which are incorporated herein by reference in their entirety.

As mentioned above, polypeptides corresponding to the polypeptides of SEQ ID NO: 2, polypeptide fragments or variants thereof can be fused or conjugated to the above-mentioned antibody moieties to increase the half-life of the polypeptides in vivo, or for use in immunoassays. Additionally, a polypeptide corresponding to SEQ ID NO: 19, may be fused or conjugated to the above antibody moieties to facilitate purification. For example one report describes a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the constant region of the light or heavy chain of a mammalian immunoglobulin. (EP 394827; Traunecker et al., Nature 331:84-86 (1988)). Polypeptides of the invention fused or conjugated to antibodies having a disulfide-linked dimer structure (due to IgG) can also bind and neutralize other molecules more efficiently than monomeric secreted proteins or fragments thereof (Fountoulakis et al. , Biochemical Journal 270: 3958-3964 (1995). In many cases, the Fc portion in the fusion protein is beneficial for therapy and diagnosis, and thus can for example improve pharmacokinetic properties (EP A232262). Alternatively, the fusion protein can be The Fc part is missing after the protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc part can hinder therapy and diagnosis. In drug development, for example, a human protein such as hIL-5 is fused to the Fc part to High throughput screening assays were performed to identify antagonists of hIL-5. (See Bennett et al., J. Mol. Rec. 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Additionally, antibodies or fragments thereof of the invention may be fused to a marker sequence, such as to a peptide, to facilitate purification. In a preferred embodiment described above, the marker amino acid sequence is a 6-histidine peptide, such as the marker provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. As described by Gentz et al., Proc. Acad. Sci. USA 86:821-824 (1989), 6-histidines allow routine purification of fusion proteins. Other peptide tags used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)), and the "flag" tag.

The invention also encompasses antibodies or fragments thereof conjugated to diagnostic or therapeutic agents. Such antibodies can be used, for example, diagnostically to monitor the development or progression of tumors, as part of clinical testing, for example, to determine the effect of a treatment regimen. Detection is facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals using various positron emission computerized tomography imaging, and non-radioactive paramagnetic metal ions . The detectable substance can be coupled or conjugated to the antibody (or fragment thereof) directly using methods known in the art, or indirectly through an intermediate such as a linker known in the art. fragment). See, eg, US Patent No. 4,741,900 for a description of metal ions that can be conjugated to antibodies, which are useful as diagnostic agents in accordance with the present invention. Suitable enzymes include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and avidin/biotin suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine, dansyl chloride or phycoerythrin; luminescent substances include, for example, luminol; bioluminescent substances such as These include luciferase, luciferin and aequorin; suitable radioactive ^substances include125I, ^131I , ^111In , ^or99Tc .

Additionally, antibodies or fragments thereof may be conjugated to therapeutic components such as cytotoxins such as cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions such as alpha-emitters such as ²¹³ Bi. A cytotoxin or cytotoxic agent includes any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincristine, vinblastine, autumn Narcissin, doxorubicin, daunorubicin, dihydroxyanthraxin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine , lidocaine, propranolol, and puromycin and their analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, dichloromethyldiethylamine, thioepa Chlorambucil, phenylalanine mustard, carmustine (BSNU) and cyclohexylnitrosourea (CCNU), cyclothosphamide, busulfan, bromomannitol, streptozotocin, mitomycin C, and dichlorodiamine cisplatin(II) (DDP), anthracyclines (such as daunorubicin, (formerly daunomycin) and doxorubicin), antibiotics (such as daunorubicin ( Actinomycin (formerly), bleomycin, mithramycin, amphifamycin (AMC)), and antimitotic agents (eg, vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response, and the therapeutic agent or drug component need not be limited to traditional chemotherapeutic agents. For example, a pharmaceutical component can be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet Derived growth factors, tissue plasminogen activator, apoptosis agents such as α-TNF, β-TNF, AIMI (see International Publication No. WO 97/33899), AIMII (see International Publication No. WO 97 /34911), Fas ligand (Takahashi et al., Immunology Research 6:1567-1574 (1994)), VEGI (see International Publication No.WO 99/23105), CD40 ligand, thrombotic (thrombotic) agent or Anti-angiogenic agents such as angiostatin or endostatin; or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), or other growth factors.

Antibodies can also be attached to solid supports, which are particularly useful in immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Methods for conjugating such therapeutic components to antibodies are well known, see eg Arnon et al., "Drug Immunodirected Monoclonal Antibodies in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.). P243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies for Drug Delivery", Controlled Drug Delivery (Second Edition), Robinson et al. (eds.), 623-53 (Marcel Dekker, Inc. 1987 ); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy", Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. (eds.), P475-506 (1985); Analysis, Results and Expected Results", Monoclonal Antibodies in Cancer Detection and Therapy, Balolwin et al., P303-16 (Science Press 1985), and Thorpe et al., "Preparation and Cytotoxicity of Antibody-Toxin Conjugates", Immuno Scientific Research 62:119-58 (1982).

Alternatively, an antibody can be conjugated to another antibody to form an antibody heteroconjugate as described by Segal in US Patent No. 4,676,980, which is hereby incorporated by reference in its entirety.

Antibodies with or without a therapeutic component conjugated thereto, administered alone or in combination with cytotoxic factors and/or cytokines, may be used in therapy.

Immunophenotyping

The antibodies of the invention are useful for immunophenotyping of cell lines and biological samples. The translation products of the genes of the invention can be used as cell-specific markers, or more particularly as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of a particular type of cell. Monoclonal antibodies directed against a specific epitope or combination of epitopes can be used to screen for marker-expressing cell populations. Various methods of screening cell populations expressing markers with monoclonal antibodies are available, including magnetic separation using antibody-coated magnetic beads, panning with antibodies attached to solid substrates (i.e., plates), and flow cytometry ( See, eg, US Patent 5,985,660; and Morrison et al., Cell 96:737-49 (1999)).

These methods are used to screen specific populations of cells such as blood cancers (ie, MRD in acute leukemia patients) and transplanted "non-self" cells to prevent graft/host disease (GVHD). Alternatively, these methods can be used to screen for the ability to proliferate and/or differentiate hematopoietic stem and progenitor cells found in human cord blood.

Antibody binding assay

Antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive or non-competitive assay systems such as Western blot analysis, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction , Gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, etc. Such assays are routine and well known in the art (see, eg, Ausubel et al., eds., 1994, General Methods in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York; incorporated herein by reference in its entirety). Some immunoassays are briefly exemplified below (but not meant to be limiting).

Immunoprecipitation methods generally involve RIPA buffer (1% NP-40 or Triton X-100, 1% deoxygenated Sodium cholate, 0.01% SDS, 0.15M Nacl, 0.01M sodium phosphate, pH 7.2, 1% Trasylαl) to lyse the cell population, add the corresponding antibody to the cell lysate, and incubate at 4°C for a period of time (such as 1-4 hours), add Protein A and/or Protein G Sepharose beads to the cell lysate, incubate at 4°C for approximately 1 hour or more, wash the beads in the lysate, and resuspend them in SDS/ in the sample buffer. The ability of the corresponding antibody to immunoprecipitate a particular antigen can be determined, for example, by Western blot analysis. Those skilled in the art will know that parameters can be modified to improve antibody binding to antigen and reduce background (eg, preclearing cell lysates with sepharose beads). For further elaboration of immunoprecipitation methods see, eg, Ausubel et al., eds., 1994, General Methods in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, at 10.16.1.

Western blot analysis generally includes preparing protein samples, electrophoresis protein samples in polyacrylamide gel (such as 8%-20% SDS-PAGE is selected according to the molecular weight of antigen), and moving protein samples from polyacrylamide gel to membrane , such as nitrocellulose, PVDF or nylon membrane, block the membrane in blocking solution (such as PBS with 3% BSA or skim milk), wash the membrane in washing buffer (such as in PBS-Tween 20) , block the membrane with a primary antibody (corresponding antibody) diluted in blocking solution, wash the membrane in washing buffer, block the membrane with a secondary antibody (which recognizes the primary antibody, such as an anti-human antibody), and the secondary antibody is diluted in solution, conjugated to an enzymatic substrate (such as horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (such as ³² P or ¹²⁵ I), the membrane is washed in the wash solution, and the presence of the antigen is detected Condition. Those skilled in the art know that parameters can be modified to increase the detected signal and reduce background interference. For a further elaboration of Western blot methods see, eg, Ausubel et al., eds., 1994, General Methods in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, at 10.8.1.

ELISAs involve preparing the antigen, coating a 96-well microtiter plate with the antigen, adding the corresponding antibody conjugated to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) to the wells, and incubating for a period of time , to detect the presence of the antigen. In ELISAs, the corresponding antibody need not necessarily be linked to a detectable compound; instead, a secondary antibody (which recognizes the primary antibody) conjugated to the detectable compound can be added to the wells. Alternatively, instead of coating the wells with antigen, the wells can be coated with antibodies. In this case, the secondary antibody conjugated to the detectable compound can be added after the addition of the corresponding antigen to the coated wells. Those skilled in the art will recognize that parameters can be modified to increase the detection signal, as will other variations of ELISAs known in the art. For further elaboration on ELISA see eg Ausubel et al. eds., 1994, General Methods in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, in 11.2.1.

The binding affinity of the antibody to the antigen and the off-rate of the antibody-antigen interaction can be determined by competitive binding assays. Competitive binding assays such as radioimmunoassays involve incubating a labeled antigen (eg ³ H or ¹²⁵ I) with the corresponding antibody in the presence of increasing amounts of unlabeled antigen and detecting antibody bound to the labeled antigen . The affinity and binding-off rate of the corresponding antibody to the specific antigen can be determined from the data obtained by Scatchard blot analysis. Competition with a second antibody can also be determined by radioimmunoassay. In this case, the antigen is incubated with the corresponding antibody conjugated to a labeled compound (such ^{as3H or125I} ) in the presence of increasing amounts of another antibody which is not labeled.

therapeutic use

The invention also relates to an antibody-based method of therapy comprising administering an antibody of the invention to an animal, preferably a mammal, more preferably a human patient, for the treatment of one or more of said diseases, dysfunctions or pathological conditions. Therapeutic compounds of the present invention include, but are not limited to, antibodies of the present invention (including fragments, analogs and derivatives thereof), and antibodies encoding the present invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies). The antibody of the present invention can be used to treat, inhibit or prevent diseases related to abnormal expression and/or activity of the polypeptide of the present invention. Dysfunctional or pathological conditions, including but not limited to any one or more of the diseases, dysfunctional or pathological conditions described herein (e.g. autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, Autoimmune cytopenias; hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg, IgA nephropathy), multiple Sclerosis, neuritis, uveitis, polyendocrine disorders, purpura (eg, Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Pane syndrome, insulin dependence Type 2 diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroiditis, hypothyroidism (ie Hashimoto's thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, recipient autoimmunity such as ( a) Graves disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderm with anti-collagen antibodies, mixed Connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, S Jogren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance for asthma or bladder fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland disorders, Vitiligo, vasculitis, post-MI, myocardial syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy, and other inflammatory, granulamatous, degenerative, and atrophic dysfunctions).

In a specific embodiment, the antibodies of the invention are used in the treatment, inhibition, prognosis, diagnosis or prevention of rheumatoid arthritis.

In another specific embodiment, the antibodies of the invention are used for the treatment, inhibition, prognosis, diagnosis or prevention of systemic lupus erythematosus.

Treatment and/or prevention of diseases, disorders or pathological conditions associated with aberrant expression and/or activity of polypeptides of the present invention includes, but is not limited to, alleviation of symptoms associated with such diseases, disorders or pathological conditions. Antibodies of the invention can also be used to target and kill cells that express Neutrokine-[alpha] on their surface, or cells that have Neutrokine-[alpha] bound on their surface. Antibodies of the invention may be provided in pharmaceutically appropriate compositions known in the art or described herein.

Methods for the therapeutic use of the antibodies of the invention include local or systemic in vivo binding of polynucleotides or polypeptides of the invention, or through direct cytotoxicity of the antibodies, such as mediated by complement (CDC) or effector cells (ADCC). Some such methods are set forth in more detail below. With the teachings herein, one skilled in the art will know how to use the antibodies of the invention for diagnosis, monitoring or therapy without undue experimentation.

Antibodies of the invention may be advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors such as IL-2, IL-3 and IL-7, e.g. to enhance effector interaction with the antibody The number or activity of cells.

Antibodies of the invention can be administered alone or in combination with other types of treatment regimens (eg, radiation therapy, chemotherapy, hormone therapy, immunotherapy, antineoplastic agents, antibiotics, and immunoglobulins). In general, it is preferred to administer a species-derived or species-reactive (in the case of antibodies) product of the same species as the patient. Thus, in a preferred embodiment, human antibodies, fragment derivatives, analogs or nucleic acids are administered to human patients for treatment or prophylaxis.

High affinity and/or potent in vivo inhibitory and/or neutralizing antibodies against polypeptides or polynucleotides of the invention are preferably used for immunoassays associated with polynucleotides or polypeptides of the invention, including fragments thereof and treatment. Such antibodies, fragments or regions preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include dissociation constants or Kd less than 5×10 ^-5 M, 10 ^-5 M, 5×10 -6 M, 10 ^-6 M, 5× ^{10 -7 M} , 10 ^-7 M, 5 × ^10-8 M, ^10-8 M, 5× ^10-9 M, ^10-9 M, 5× ^10-10 M, ^10-10 M, 5× ^10-11 M, ^10-11 M, 5×10 ^-12M , ^10-12M , 5× ^10-13M , ^10-13M , 5× ^10-14M , ^10-14M , 5× ^10-15M , and ^10-15M .

Gene therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof are administered to treat, inhibit or prevent diseases or dysfunctions associated with abnormal expression and/or activity of the polypeptides of the present invention by gene therapy. Gene therapy refers to treatment by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates the therapeutic effect.

Any suitable method of gene therapy known in the art may be used in accordance with the present invention. For example, the method described below.

For general research on gene therapy see Goldspiel et al., Clinical Pharmacology 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Analytical Research in Toxicology 32:573-596 (1993 ); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Biochemical Analytical Research 62:191-217 (1993); May, TIB TECH 11(5):155-215 (1993). Recombinant DNA techniques known in the art that can be used are described in Ausubel et al. (eds.), General Methods in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, A Laboratory Manual of Gene Transfer and Expression, Stockton Press, NY (1990). stated.

In a preferred embodiment, the compound comprises a nucleic acid sequence encoding an antibody which is part of an expression vector for expressing the antibody or fragment thereof or chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, said promoter being inducible or constitutive, and optionally tissue-specific. In another specific embodiment, nucleic acid molecules are used wherein the antibody coding sequence and any other desired sequences are flanked by regions that initiate homologous recombination at desired sites in the genome, thus providing for intrachromosomal expression of the antibody coding nucleic acid (Koller and Smithies, Proceedings of the National Academy of Sciences 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single-chain antibody; alternatively, a nucleic acid Sequences include sequences encoding antibody heavy and light chains or fragments thereof.

The delivery of nucleic acid to the patient can be direct or indirect. The direct way is that the patient is directly exposed to the nucleic acid or the carrier carrying the nucleic acid. The indirect way is that the cells are first transformed with the nucleic acid in vitro and then transplanted into the patient. These two approaches are known as in vivo or in vivo-derived gene therapy, respectively.

In a specific embodiment, the nucleic acid sequence is administered directly in vivo, where its expression produces the encoded product. This can be done by various methods known in the art, for example, constructing them as part of appropriate nucleic acid expression vectors and administering them to make them intracellular, for example by using defective or weakened retroviruses or other Infection with viral vectors (see US Patent No. 4980286), either by direct injection of naked DNA, or using microparticle bombardment (eg, gene gun; Biolistic, Dupont), or coating with lipid or cell surface receptors or transfection agents, Encapsulation with liposomes, microparticles or microcapsules, or administration via linking to peptides known to enter the nucleus, for receptor-mediated endocytosis by linking administration to ligands (see e.g. Wu and Wu, J. Biochem. 262: 4429-4432 (1987)) (which can be used to target cell types that specifically express the receptor). In another embodiment, nucleic acid-ligand complexes can be formed, wherein the ligand comprises a fused viral peptide to disrupt endosomes and protect nucleic acids from lysosomal degradation. In another embodiment, nucleic acids can be targeted in vivo to cells for specific uptake and expression via targeting-specific receptors (see, e.g., PCT publications WO92/06180; WO92/22635; WO92/20316; WO93/14188; WO923/ 20221). Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression (Koller and Smithies, Proc. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989) ).

In a specific embodiment, a viral vector comprising a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used (see Miller et al., Methods in Enzymology 217:581-599 (1993). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration of lambda host cell DNA. The encoded gene therapy The nucleic acid sequence of the antibody used in cloning lambda in one or more vectors that facilitate gene delivery to the patient. A more detailed description of retroviruses is found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to generate stem cells that are more resistant to chemotherapy. Other examples of the use of retroviral vectors in gene therapy are found in Clowes et al., J. Clin. Invest. 93:644-651( 1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Gene Therapy in Humans 4: 129-141 (1993); and Grossman and Wilson, General Perspectives in Genetics and Development 3: 110-114 ( 1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly useful delivery vehicles for gene delivery to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, causing mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect undifferentiated cells. Kozansky and Wilson, General Perspectives in Genetics and Development 3: 499-503 (1993) propose an adenovirus-based approach to gene therapy. Bout et al., Gene Therapy in Humans 5:3-10 (1994) describe the use of adenoviral vectors to transfer genes to the respiratory epithelium of asthmatic monkeys. Additional references to the use of adenoviruses in gene therapy are found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993) ; PCT Publication WO 94/12649; and Wang et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, an adenoviral vector is used.

Adeno-associated virus (AAV) has been used in gene therapy for this purpose (Walsh et al., PNAS 204:289-300 (1993); US Patent No. 5436146).

Another approach to gene therapy involves transferring genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, methods of transfer involve the transfer of a detectable label to the cells. The treated cells expressing the transgene are then isolated. These cells are then administered to the patient.

In this embodiment, the nucleic acid is introduced into the cells prior to administration of the resulting recombinant cells in vivo. Such introduction can be performed by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with viral or phage vectors containing nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, minicells Mediated gene transfer, spheroplast fusion, etc. Many methods for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Beht, Methods in Enzymology 217: 599-618 (1993); Cohen et al., Methods in Enzymology 217: 618-644 (1993); Clinical Drug Therapy 29:69-92m (1985), these methods can be used according to the present invention, and do not destroy the basic development of recipient cell and physiological function.Use method should stably move nucleic acid to cell, so that nucleic acid can be expressed by cell and preferably can be obtained by its Cell progeny inheritance and expression.

The resulting recombinant cells can be administered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem or progenitor cells) are preferably administered intravenously. The number of cells used depends on the desired effect, the state of the patient, etc., and can be determined by one skilled in the art.

The cells into which the nucleic acid is introduced for gene therapy encompass any desired appropriate cell type, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, myocytes, hepatocytes, blood cells such as T lymphocytes Cells, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, especially hematopoietic stem or progenitor cells, as obtained from bone marrow . Cells from umbilical cord blood, peripheral blood, fetal liver, etc.

In preferred embodiments, the cells used for gene therapy are the patient's own.

In embodiments where recombinant cells are used for gene therapy, nucleic acid sequences encoding antibodies are introduced into the cells so that they can be expressed by the cells or cell progeny, and the recombinant cells are then administered in vivo for therapy. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells that can be isolated and maintained in vitro can be used according to this embodiment of the invention (see, e.g., PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Methods in Cell Biology 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986).

In specific embodiments, nucleic acids introduced for gene therapy comprise an inducible promoter operably linked to the coding region, whereby expression of the nucleic acid can be controlled by controlling the presence or absence of an appropriate transcriptional inducer.

Evidence of therapeutic or prophylactic activity

The compounds or pharmaceutical compositions of the present invention are preferably first tested in vitro and then tested in vivo for the desired therapeutic or prophylactic activity before being used in humans. For example, in vitro assays demonstrating therapeutic or prophylactic activity of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on a cell line or patient tissue sample can be determined using methods known in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the present invention, in vitro assays that can be used to determine whether a specific compound is administered are indicated, including in vitro cell culture assays, wherein a patient tissue sample is grown in culture, exposed to or administered a compound, and the effect of such compound on the tissue sample is observed. role.

Methods and compositions for therapeutic and/or prophylactic administration

The present invention provides methods for achieving therapeutic, inhibitory and prophylactic purposes by administering an effective amount of the compound or pharmaceutical composition of the present invention, preferably the antibody of the present invention, to the subject. In a preferred embodiment, the compound is substantially purified (eg, substantially free of substances that limit its action or produce undesired side effects). The subject to be treated is preferably an animal, including but not limited to cows, pigs, horses, chickens, cats, dogs, etc., preferably a mammal, and most preferably a human.

When the compound comprises a nucleic acid or an immunoglobulin, formulations and methods of administration may be used as described above; additional suitable formulations and routes of administration may be selected from those described below.

Various delivery systems are known and can be used to administer the compounds of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, Biochemical Journal 262: 4429-4432 (1987)), Construction of nucleic acids as part of retroviruses or other vectors, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, sublingual, intranasal, epidermal or oral. The compounds or compositions may be administered by conventional routes, such as by fusion or bolus injection, absorbed through the epithelium or at the junction of mucosa and skin (eg, oral mucosa, rectal and small intestinal mucosa, etc.), and may be co-administered with other biologically active agents. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be introduced into the central nervous system by any suitable route, including intravenous and intraarterial injection; intravenous injection is facilitated by intravenous catheters, such as adsorption to a reservoir such as an Ommaya reservoir. Pulmonary administration can also be used, such as with an inhaler or nebulizer, and formulations with aerosols.

In a specific embodiment, it is desirable to administer the pharmaceutical compound or composition of the invention locally at the site in need of treatment; this may be by, for example, local infusion during surgery, topical application, such as with a wound dressing after surgery, by injection , applied by catheter, by suppository, or by implant, said implant being porous, non-porous, or gelatinous, comprising membranes, such as sialastic membranes, or fibers. Preferably, when administering proteins of the invention, including antibodies, care must be taken to use substances that are not absorbed by the protein.

In another embodiment, the compound or composition can be delivered in vesicles, especially liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in Infectious Disease and Cancer Therapy, vol. Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, supra, pp. 317-327).

In another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N . Engl. J. Med. 321:574 (1989)). In another embodiment, polymers can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Design and Manufacture, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol, Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985) ; During et al., Neurol Res. 25:351 (1989); Howanol et al., Neurosurg J. 71:105 (1989)). In another embodiment, the controlled release system can be placed near the target of treatment, namely the brain, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138( 1984).

Other controlled release systems are described by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to initiate expression of the protein it encodes by constructing it as part of an appropriate nucleic acid expression vector and administering it such that Become part of the cell, for example by using retroviral vectors (see US Patent No. 4980286), or by direct injection, or by using particle bombardment (eg gene gun; Biolistic, Dupont), or by using lipid or cell surface Coating with transfectants or transfection agents, or administration via linkage to homeobox-like peptides known to enter the nucleus (see, eg, Joliot et al., Proc. Acad. Sci. USA 88:1864-1868 (1991)) and the like. Alternatively, nucleic acids can be introduced into cells by homologous recombination and incorporated into host cell DNA for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically suitable carrier. In a specific embodiment, the term "pharmaceutically suitable" refers to an article approved by a federal or governmental agency or listed by a US drug regulatory agency, or otherwise generally recognized as acceptable for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and dextrose and glycerol solutions can also be employed as liquid carriers. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, lime, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk , glycerin, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Suitable pharmaceutical carriers are described, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of the compound, preferably in purified form, and an appropriate amount of carrier to provide proper dosage form for administration to a patient.

In a preferred embodiment, the composition is formulated according to conventional methods of preparing pharmaceuticals suitable for intravenous administration to humans. Typically, compositions for intravenous administration are sterile isotonic solutions. The composition, if desired, can also include stabilizers and a local anesthetic, such as lignocaine, to relieve pain at the site of injection. Generally, the ingredients are presented singly or in unit dosage mixes, eg, as anhydrous lyophilized powders or anhydrous concentrates in hermetically sealed containers, such as ampoules or sachets indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be used to mix the ingredients prior to administration.

The compounds of the present invention can be formulated as neutral or salt forms. Suitable salts of pharmaceuticals include those with cations such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., or with anions such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isohydroxide, Salts formed by the anions of propylamine, triethylamine, 2-ethylaminoethanol, histamine, procaine, etc.

The amount of a compound of the invention effective in treating, inhibiting and preventing a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. The actual dosage employed in the formulation will also depend on the route of administration, the severity of the disease, and should be determined by the physician's diagnosis and the patient's condition. Effective doses can be determined from dose-response curves generated in vitro or in animal model test systems.

As for the antibody, the dosage administered to the patient is between 0.1-100 mg/kg body weight, more preferably, between 1-10 mg/kg body weight. In general, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to the foreign polypeptide. Thus, fewer doses of human antibodies can be administered, and the frequency of administration can also be longer. In addition, the dose and frequency of administration of the antibodies of the present invention can be reduced by modifying the antibody, such as lipidation, to enhance antibody absorption and tissue permeability (eg, into the brain).

The invention also provides a pharmaceutical pack or kit comprising one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Optionally associated with such a container is a notice by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product, the notice reflecting the authorization to manufacture, use or market the biological product or drug for human administration.

Diagnostics and Imaging

Labeled antibodies that specifically bind to the corresponding peptides, and derivatives and analogs thereof, can be used for diagnostically detecting, diagnosing or monitoring diseases and/or dysfunctions related to abnormal expression and/or activity of the polypeptides of the present invention. The present invention provides a method for detecting the abnormal expression of the corresponding polypeptide, comprising (a) analyzing the expression of the corresponding polypeptide in individual cells or body fluids with one or more antibodies specific to the corresponding polypeptide, and (b) measuring the expression level of the gene An increase or decrease in the gene expression level of the assayed polypeptide compared to a standard gene expression level, compared to a standard gene expression level, indicates abnormal expression.

The present invention provides a diagnostic assay for diagnosing dysfunction, comprising (a) analyzing the expression of the corresponding polypeptide in cells or body fluids of an individual using one or more antibodies specific for the corresponding polypeptide, and (b) analyzing the expression level of the gene An increase or decrease in the expression level of the analyzed polypeptide gene compared to the standard expression level, compared to the standard expression level, indicates a specific dysfunction. In the case of cancer, the presence of relatively high amounts of transcripts in individual biopsies can indicate The predisposition to disease occurrence may provide a means of detecting the disease before true clinical symptoms appear, and a more certain diagnosis may allow health practitioners to apply preventive measures or aggressive treatment earlier, thereby preventing the onset or further progression of cancer.

The antibodies of the invention can be used to analyze protein levels in biological samples using conventional immunohistological methods known to those skilled in the art (see, e.g., Jalkanen et al., J. Cell Biology 101:976-985 (1985); Jalkanen et al., Cell Journal of Biology 105:3087-3096 (1987)). Other antibody-based methods for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Appropriate labels for antibody analysis are known in the art, including enzyme labels such as glucose oxidase; radioactive isotopes such as iodine ( ¹³¹ I, ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S) tritium ( ³ H) , indium ( ^115m In, ^113m In, ¹¹² In, ¹¹¹ In), and technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), Xenon ( ¹³⁸ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr , ¹⁰⁵ Rh, ⁹⁷ Ru; luminescent labels such as Luminol; and fluorescent labels such as fluorescein, and rhodamine, and biotin.

Antibodies of the invention can be labeled by methods known in the art. Such methods include, but are not limited to, the use of bifunctional conjugates (see, eg, U.S. Patent Nos. 5,756,065; 5,714,631; 5,996,239; 5,652,361; incorporated by reference).

One embodiment of the present invention is to detect and diagnose diseases or dysfunctions associated with abnormal expression of corresponding polypeptides in animals, preferably mammals, more preferably humans. In one embodiment, the diagnostic method comprises (a) administering to the diagnostic subject an effective amount of a labeled molecule that specifically binds to the corresponding polypeptide, such as by parenteral, subcutaneous or intraperitoneal administration; (b) waiting for a period of time after administration, preferentially concentrate labeled molecules at sites of polypeptide expression (and eliminate unlabeled molecules); (c) determine background levels; and (d) detect labeled molecules in the diagnostic subject such that detection of labeled molecules above background levels indicates a diagnosis The subject has a specific disease or dysfunction related to abnormal expression of the corresponding polypeptide. Background levels can be determined by a variety of methods, including comparing the amount of detected labeled molecule to a standard value previously established in a particular system. As described herein, a specific embodiment of the invention is the quantitative or qualitative enrichment of cells of the B cell lineage or cells of the monocytic lineage using the antibodies of the invention.

As also described herein, the antibodies of the invention are useful for the treatment, diagnosis or prophylaxis of individuals with immunodeficiency, in a specific embodiment the antibodies of the invention are for the treatment, diagnosis and/or prophylaxis of individuals with CVID or subtypes thereof individual. In another embodiment, the antibodies of the invention are used in the diagnosis, prophylaxis, and treatment of diseases characterized by defective production of serum immunoglobulins, recurrent infections, and/or immune system dysfunction.

Also as described herein, the antibodies of the invention are useful in the treatment, diagnosis or prognosis of individuals with an autoimmune disease or disorder. In a specific embodiment, the antibodies of the invention are used in the treatment, diagnosis and/or prognosis of individuals with systemic lupus erythematosus or a subtype of the disease. In another specific embodiment, the antibodies of the invention are used in the treatment, diagnosis and/or prognosis of individuals with rheumatoid arthritis or a subtype thereof.

It is understood in the art that the size of the subject being treated and the imaging system used will determine the number of imaging components required to produce a diagnostic image. In the case of radioisotopic components, the normal amount of radioactivity injected is between about 5-20 ^mCi99 mTc in humans. The labeled antibody or antibody fragment then accumulates preferentially in cells containing the specific protein. In vivo tumor imaging is found in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Fragments thereof" (Chapter 13 in Tumor Imaging: Radiochemical Detection of Cancer, eds. SW Burchiel and BARhocles, Masson Publishing Inc. (1982)) mentioned.

Depending on a number of variables, including the type of label used and the mode of administration, the post-administration interval is 6-48 hours, or 6-24 hours, or 6-12 hours, the interval being such that the labeled molecule is preferentially concentrated at the target site, and the Unbound labeled molecules were cleared to background levels. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, the disease or disorder is monitored by repeated diagnosis of the disease, eg, one month after initial diagnosis, six months after, one year after, etc.

The presence or absence of a marker molecule in a patient can be detected by in vivo scanning methods known in the art. These methods depend on the type of marker used. The skilled artisan can determine the appropriate method for detecting a particular marker. Methods and instruments that may be used in the diagnostic methods of the present invention include, but are not limited to, computerized tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance (MRI) and ultrasound.

In a specific embodiment, the molecule is labeled with a radioactive isotope and detected in a patient using radiation-responsive surgical instruments (Thurston et al., US Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence response scanner, in another embodiment the molecule is labeled with a positron-emitting metal and scanned in the patient using positron emission tomography detection. In another embodiment, the molecule is labeled with a paramagnetic marker and detected in a patient using nuclear magnetic resonance (MRI).

Reagent test kit

The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises one or more containers containing an antibody of the invention, preferably a purified antibody. In a specific embodiment, the kit of the invention comprises a substantially isolated polypeptide comprising The antibodies in the kit are specific for immunoreactive epitopes. Preferably, the kit of the present invention further comprises a control antibody that does not react with the corresponding polypeptide. In another specific embodiment, the kit of the invention comprises two or more antibodies (monoclonal and/or polyclonal) recognizing the same and/or different sequences or regions of the polypeptide of the invention. In another specific embodiment, the kit of the invention comprises means for detecting the binding of the antibody to the corresponding polypeptide (e.g. the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound). The compound, or a second antibody that recognizes the first antibody, can be conjugated to a detectable substrate).

In another specific embodiment of the invention, the kit is a diagnostic kit for screening sera containing antibodies specific against proliferative and/or cancer polynucleotides and polypeptides. Such kits may include control antibodies that do not react with the corresponding polypeptide. Such kits may include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one antibody raised against the polypeptide antigen. Additionally, such kits include means to detect binding of the antibody to the antigen (eg, the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine detectable by flow cytometry). In specific embodiments, kits may include recombinantly produced or chemically synthesized polypeptide antigens. The polypeptide antigens of the kit can also be attached to a solid support.

In a more specific embodiment, the detection means of the above kit includes a solid support to which the polypeptide antigen is attached, and this kit may also include an unattached receptor-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen is detectable by binding of the receptor-labeled antibody.

In another embodiment, the invention includes a diagnostic kit for screening sera containing polypeptide antigens of the invention. The diagnostic kit comprises a substantially isolated antibody specifically immunoreactive with a polynucleotide or polypeptide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support, and in a specific embodiment, the antibody may be a monoclonal antibody. The detection means in the kit may include a second labeled monoclonal antibody. Alternatively, the assay can include a labeled competing antigen.

In a diagnostic configuration, the test serum is reacted with a solid phase reagent having surface-bound antigen obtained by the method of the invention. After the antibody to the specific antigen is bound to the reagent and the bound serum components are removed by washing, the reagent is reacted with the receptor-labeled anti-human antibody, and the receptor-to-reagent binding ratio is the amount of antibody against the antigen on the solid support. The reagent is washed again to remove unbound labeled antibody and to determine the amount of receptor associated with the reagent. In particular, the receptor is an enzyme that is detected by incubating the solid phase in the presence of an appropriate fluorescent, luminescent or colorimetric substrate (Sigma, St. Louis, Mo).

The solid surface reagents in the above assays are prepared by methods known in the art for attaching proteins to solid supports, such as polymeric beads, dip strips, 96-well plates or membrane filters. These attachment methods generally include non-specific adsorption of proteins to supports, or chemically reactive genes that covalently attach proteins to solid supports through free amino groups, such as active carboxyl, hydroxyl or aldehyde groups. Alternatively, streptavidin-coated plates can be used in conjugation with biotinylated antigen.

Accordingly, the present invention provides assay systems or kits for performing such diagnostic methods. The kit generally includes a support having a surface-bound recombinant antigen, and a receptor-labeled anti-human antibody to detect the surface-bound anti-antigen antibody.

The invention also relates to antibodies that are agonists or antagonists of the polypeptides of the invention. For example, the invention includes antibodies that partially or fully disrupt the receptor/ligand interaction with the polypeptides of the invention, including both receptor-specific and ligand-specific antibodies. Wherein the receptor-specific antibody does not prevent ligand binding but prevents receptor activation. Receptor activation (ie, signaling) can be determined by methods described herein or known in the art. Receptor-specific antibodies that prevent ligand binding and receptor activation are also included. Also included are neutral antibodies that bind a ligand and prevent binding of the ligand to the receptor, antibodies that bind the ligand thereby preventing activation of the receptor but do not prevent binding of the ligand to the receptor, and antibodies that activate the receptor. These antibodies can act as agonists, affecting all or part of the biological activity through ligand-mediated receptor activation. Antibodies may specifically act as agonists or antagonists of biological activities comprising the specific activities described herein. Also included are antibodies that bind Neutrokine-alpha and/or Neutrokine-alphaSV whether or not Neutrokine-alpha or Neutrokine-alphaSV binds to the Neutrokine-alpha receptor. These antibodies act as Neutrokine-α and/or Neutrokine-αSV agonists and, in the presence of these antibodies, increase cell proliferation in response to binding of Neutrokine-α and/or Neutrokine-αSV to the Neutrokine-α receptor. The above antibody agonists can be produced by methods known in the art. See eg WO96/40281; US Patent 5811097; Deng, B. et al. Blood 92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res. 58(16):3668-3678 (1998); Harrop, J.A. et al., J. Immunol. 161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, D.Y. et al., J. Immunol. 160(7) : 3170-3179 (1998); Prat, M. et al., J. Cell Sci. 111 (Pt2): 237-247 (1998); Ptard, V. et al., J. Immunol. Methods 205(2): 177-190 (1997) ; Lautard, J. et al., Cytokine 9(4): 233-241 (1997); Carlson, N.G. et al., J. Biochem. 272(17): 11295-11301 (1997); Taryman, R.E. et al., Neuron 14 ( 4): 755-762 (1995); Muller, Y.A. et al., Structure 6(9): 1153-1167 (1998); Bartunek, P. et al., Cytokine 8(1): 14-20 (1996) ( The entire text of said document is incorporated by reference).

At least 14 monoclonal antibodies have been produced against Neutrokine-alpha, these monoclonal antibodies are called 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12, 16B4, 3D4, 16C9, 13D5, 15C10, and 12C5. Preliminary analysis of these antibodies showed binding to Neutrokine-α both in Western blot analysis and when Neutrokine-α protein was bound to ELISA plates. However, further analysis of the 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12 and 16B4 antibodies revealed that only the 12D6, 9B6, 2E5, 10G12, 9A5 and 11G12 antibodies bound the membrane-bound form of Neutrokine-α. Therefore, it has been determined that the subtype of the monoclonal antibody against Neutrokine-α produced only binds to the membrane-bound form of Neutrokine-α (that is, this subtype does not bind to the amino acid corresponding to the 134-285 positions of SEQ ID NO: 2). lytic form of Neutrokine-alpha), as described herein, is restricted to expression on monocytes and dendritic cells.

It has now been found that the 9B6 antibody specifically binds to the membrane-bound form of Neutrokine-α, but not to the soluble form of Neutrokine-α.

The epitope map of antibody 9B6 shows that this antibody specifically binds to the amino acid sequence contained in the Ser171-Phe194 amino acid residues of SEQ ID NO:2. More particularly, the epitope map indicates that antibody 9B6 specifically binds a peptide comprising amino acid residues Lys 173-Lys 188 of SEQ ID NO:2.

In contrast, antibodies 16C9 and 15C10 were found to bind a soluble form of Neutrokine-α (ie, amino acids 134-285 of SEQ ID NO: 2) and inhibit Neutrokine-α-mediated B cell proliferation. See eg Example 10, the 15C10 antibody has been found to inhibit the binding of Neutrokine-alpha to its receptor. The epitope map of antibody 15C10 has shown that this antibody specifically binds to the amino acid sequence contained in Glu223-Tyr246 amino acid residues of SEQ ID NO:2. More particularly, the epitope map indicates that antibody 15C10 specifically binds a peptide comprising amino acid residues Val 227-Asn 242 of SEQ ID NO:2. Antibody 15C10 also specifically binds to a peptide comprising amino acid residues Phe 230-Cys245 of SEQ ID NO:2.

As mentioned above, anti-Neutrokine-α monoclonal antibodies have been prepared. The hybridomas producing the 9B6 and 15C10 antibodies were deposited with the ATCC under the accession numbers PTA-1158 and PTA-1159, respectively. In one embodiment, the antibody of the present invention has one or more of the same biological properties as the antibody secreted by the hybridoma cell line with deposit number PTA-1158 or PTA-1159. "Biological property" refers to the in vitro or in vivo activity or property of the antibody, such as the ability to bind Neutrokine-α (such as the polypeptide of SEQ ID NO: 2, the mature form of Neutrokine-α, the membrane-bound form of Neutrokine-α, the soluble form Neutrokine-α (134-285 amino acids of SEQ ID NO: 2), and the antigen region and/or epitope region of Neutrokine-α), basically block the ability of Neutrokine-α to bind to its receptor, or block Neutrokine-α - Ability to alpha-mediated biological activity (such as stimulation of B cell proliferation and immune protein production). Optionally, an antibody of the invention binds to the same epitope as at least one antibody specified. Such epitope binding can be routinely determined by methods known in the art.

Thus, in one embodiment, the invention provides antibodies that specifically bind the membrane-bound form and not the soluble form of Neutrokine-[alpha]. Uses of these antibodies include, but are not limited to, as diagnostic probes to identify and/or isolate monocytic cell lines expressing the membrane-bound form of Neutrokine-alpha. For example, expression of the membrane-bound form of Neutrokine-alpha is increased on activated monocytes, thus, antibodies contemplated by the invention can be used to detect and/or determine the level of activated monocytes. Alternatively, antibodies that bind only the membrane-bound form of Neutrokine-α can be used to target the toxin to tumorigenic, pre-tumorigenic, and/or other cells expressing the membrane-bound form of Neutrokine-α (such as monocytes and dendritic cells). cell).

In another embodiment, the antibody of the present invention specifically binds only the soluble form of Neutrokine-α (amino acids 134-285 of SEQ ID NO: 2). Uses of these antibodies include, but are not limited to, use as diagnostic probes to analyze soluble forms of Neutrokine-α in biological samples, and as therapeutic agents to direct toxins to cells expressing Neutrokine-α receptors (such as B cells), and /or reduce or block Neutrokine-α-mediated biological activity in vitro or in vivo (such as stimulation of B cell proliferation and/or immunoglobulin production).

The present invention also provides antibodies that specifically bind membrane-bound and soluble forms of Neutrokine-alpha.

As mentioned above, the present invention encompasses antibodies that inhibit or reduce the ability of Neutrokine-α and/or Neutrokine-αSV to bind to its receptors in vivo and/or in vitro, and in a specific embodiment, the antibodies of the present invention inhibit or reduce Antibodies for the ability of Neutrokine-α and/or Neutrokine-αSV to bind to its receptors in vitro, in another specific embodiment, the antibodies of the present invention inhibit or reduce the ability of Neutrokine-α and/or Neutrokine-αSV to bind to their receptors in vivo Antibodies for the body's ability. Such inhibition can be assayed using methods described herein or known in the art.

The present invention also encompasses antibodies that specifically bind Neutrokine-α and/or Neutrokine-αSV, but do not inhibit the ability of Neutrokine-α and/or Neutrokine-αSV to bind its receptor in vitro and/or in vivo. In a specific embodiment, the antibodies of the invention do not inhibit or reduce the ability of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV to bind to its receptor in vitro. In another specific embodiment, the antibodies of the invention do not inhibit or reduce the ability of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV to bind their receptors in vivo.

As noted above, the present invention encompasses antibodies that inhibit or reduce Neutrokine-α and/or Neutrokine-αSV-mediated biological activity in vitro and/or in vivo. In a specific embodiment, the antibodies of the invention inhibit or reduce Neutrokine-[alpha] and/or Neutrokine-[alpha]SV-mediated B cell proliferation in vitro. Such inhibition can be assayed by conventional methods of modifying B cell proliferation described herein or known in the art. In another specific embodiment, the antibodies of the invention inhibit or reduce Neutrokine-α and/or Neutrokine-αSV mediated B cell proliferation in vivo. In a specific embodiment, the antibody of the invention is 15C10, or a humanized form thereof. In another preferred specific embodiment, the antibody is 16C9, or a humanized form thereof. Thus, in a specific embodiment of the invention, the 16C9 and/or 15C10 antibodies, or humanized forms thereof, are used to bind soluble forms of Neutrokine-α and/or Neutrokine-αSV, and/or agonists and/or antagonists thereof agents that inhibit (partially or completely) B cell proliferation.

Alternatively, the present invention also covers specific binding to Neutrokine-α and/or Neutrokine-αSV, but does not inhibit or reduce Neutrokine-α and/or Neutrokine-αSV-mediated biological activity in vitro and/or in vivo (such as stimulating B cell proliferation) antibodies. In a specific embodiment, the antibodies of the invention do not inhibit or reduce Neutrokine-α and/or Neutrokine-αSV-mediated biological activity in vitro. In another specific embodiment, the antibodies of the invention do not inhibit or reduce Neutrokine-α and/or Neutrokine-αSV-mediated biological activity in vivo. In a specific embodiment, the antibody of the invention is 9B6, or a humanized form thereof.

As noted above, the invention encompasses antibodies that specifically bind in vitro and/or in vivo to the same epitope as at least one antibody specified.

In a specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in Ser 171-Phe194 amino acid residues of SEQ ID NO: 2 in vitro. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in Ser171-Phe194 amino acid residues of SEQ ID NO:2 in vivo. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in Lys173-Lys188 amino acid residues of SEQ ID NO: 2 in vitro. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in Lys173-Lys188 amino acid residues of SEQ ID NO: 2 in vivo.

In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in the Glu223-Tyr246 amino acid residues of SEQ ID NO: 2 in vitro. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in Glu 223-Tyr 246 amino acid residues of SEQ ID NO: 2 in vivo. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in the Val227-Asn242 amino acid residues of SEQ ID NO: 2 in vitro. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in the Val227-Asn242 amino acid residues of SEQ ID NO: 2 in vivo. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in the Phe 230-Cys245 amino acid base of SEQ ID NO: 2 in vitro. In another specific embodiment, the antibody of the present invention specifically binds to the amino acid sequence contained in the Phe230-Cys245 amino acid residues of SEQ ID NO: 2 in vivo.

The present invention also provides the competitive inhibition of the 9B6 monoclonal antibody produced by the preserved PTA-1159 and the polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2, more preferably the amino acid sequence of the Ser171-Phe194 residue of SEQ ID NO: 2 Peptide-binding antibodies. Competitive inhibition can be determined by any method known in the art, eg, using the competitive binding assays described herein. In a preferred embodiment, the antibody competitively inhibits at least 95%, 90%, 85% of the binding of the 9B6 monoclonal antibody to the polypeptide of SEQ ID NO: 2, preferably the polypeptide having the Ser171-Phe194 amino acid sequence of SEQ ID NO: 2 %, 80%, 75%, 70%, 60%, 50%.

The present invention also provides a 15C10 monoclonal antibody that competitively inhibits the production of the preserved PTA-1158 hybridoma, and the polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2, more preferably Glu223-Try246 of SEQ ID NO: 2 An antibody that binds to a polypeptide of amino acid sequence. In a preferred embodiment, the antibody-competitive inhibitory 15C10 monoclonal antibody binds to the polypeptide of SEQ ID NO: 2, preferably the polypeptide having the Glu223-Tyr246 amino acid sequence of SEQ ID NO: 2 at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%.

Additional embodiments of the invention relate to the 9B6 antibody and hybridoma cell lines expressing the antibody. The hybridoma cell line expressing 9B6 antibody was deposited in ATCC on January 7, 2000 with the accession number PTA-1159. In a preferred embodiment, antibody 9B6 is humanized.

Additional embodiments of the invention relate to the 15C10 antibody and hybridoma cell lines expressing the same. The hybridoma cell line expressing antibody 15C10 was deposited in ATCC on January 7, 2000, with the accession number PTA-1158. In a preferred embodiment, antibody 15C10 is humanized.

In a specific embodiment, the specific antibodies described above are humanized using methods described herein or known in the art and then used as therapeutic agents.

In another specific embodiment, any of the above antibodies is used in soluble form.

In another specific embodiment, any of the above antibodies is conjugated to a toxin or a label (as described above). This conjugated antibody is used to kill specific cell populations, or to quantify specific cell populations. In a preferred embodiment, such conjugated antibodies are used to kill B cells expressing the Neutrokine-alpha receptor on their surface. In another preferred embodiment, such conjugated antibodies are used to quantify B cells expressing the Neutrokine-alpha receptor on their surface.

In another specific embodiment, any of the above antibodies is conjugated to a toxin or a label (as described above). Such conjugated antibodies are used to kill or quantify specific cell populations. In a preferred embodiment, such conjugated antibodies are used to kill monocytes expressing the membrane-bound form of Neutrokine-[alpha]. In another preferred embodiment, such conjugated antibodies are used to quantify monocytes expressing a membrane-bound form of Neutrokine-[alpha].

Antibodies of the invention also have therapeutic and/or prophylactic uses, including but not limited to activating monocytes or blocking monocyte activation and/or killing monocyte cell lines expressing a membrane-bound form of Neutrokine-α (such as the treatment, prevention and/or diagnosis of myeloid leukemia, monocytic-based leukemia and lymphoma, mononucleosis, monocytopenia, rheumatoid arthritis, and other cell-related disease or pathological state). In a specific embodiment, the antibodies of the invention fix complement. In other specific embodiments, antibodies of the invention (or fragments thereof) bind to heterologous polypeptides or nucleic acids (e.g., toxins, such as compounds that bind and activate endogenous cytotoxic effector systems, and radioisotopes; and cytotoxic drugs ).

In another embodiment, one or more monoclonal antibodies that recognize or bind Neutrokine-alpha and/or muteins thereof but do not recognize or bind Neutrokine-alphaSV and/or muteins thereof are produced. In a related embodiment, one or more monoclonal antibodies are produced wherein they recognize or bind Neutrokine-α and/or muteins thereof, but do not recognize or bind Neutrokine-α and/or muteins thereof.

As noted above, antibodies to Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the present invention can be used to generate anti-idiotypic antibodies that "mimic" Neutrokine-alpha using methods well known to those skilled in the art (see, e.g., Greenspan & Bona, FASEB Journal 7 (5): 437-444 (1989), and Nissionff, Journal of Immunology 147 (8): 2429-2438 (1991).For example, binding Neutrokine-α and/or Neutrokine-αSV and competitive inhibition of Neutrokine-α α and/or Neutrokine-αSV multimers and/or ligand-conjugated antibodies that can be used to generate anti-idiotypic antibodies that "mimic" Neutrokine-αTNF multimerization and/or binding structures, thereby binding and neutralizing Neutrokine -α or Neutrokine-αSV and/or its ligand. This neutralizing anti-idiotypic antibody or its Fab fragment can be used in a therapeutic regimen to neutralize the Neutrokine-α ligand. For example, this anti-idiotypic antibody can be used for Bind to Neutrokine-α and/or Neutrokine-αSV, or bind to Neutrokine-α and/or Neutrokine-αSV receptors on the surface of B cells, thereby blocking Neutrokine-α and/or Neutrokine-αSV-mediated B cell activation, proliferation and/or differentiation.

Diagnosis of diseases related to the immune system

Neutrokine-α in kidney, lung, peripheral blood leukocytes, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages, and special cells of umbilical cord blood tissue and monocyte lineage Express. In addition, Neutrokine-αSV is expressed in naive dendritic cells. In addition, Neutrokine-α is expressed on the cell surface of the following non-hematopoietic tumor cell lines: colon cancer HCT 116 (ATCC deposit No. CCL-247) and HT-29 (ATCC deposit No. HTB-38); Cancer Caco-2 (ATCC preservation number NO.HTB-37), COLO 201 (ATCC preservation number NO.CCL-224) and WiDr (ATCC preservation number NO.CCL-218); breast cancer MDA-MB-231 (ATCC preservation number No. HTB-26); bladder squamous cell carcinoma SCaBER (ATCC No. HTB-3); bladder cancer HT-1197 (ATCC No. CRL-1473); kidney cancer A-498 (ATCC No. NO. HTB-44); Caki-1 (ATCC deposit number NO.HTB-46), and Caki-2 (ATCC deposit number NO.HTG-47); kidney Wilms tumor SK-NEP-1 (ATCC deposit number NO.HTB- 48); and pancreatic cancer Hs766T (ATCC Deposit No. HTB-134); MIA PaCa-2 (ATCC Deposit No. CRL-1420) and SU.86.86 (ATCC Deposit No. CRL-1837). For many diseases related to the immune system, the change (increase or decrease) in the expression level of Neutrokine-α and/or Neutrokine-αSV compared with the standard level can be detected in the immune system tissue or "Standard" Neutrokine-α and/or Neutrokine-αSV gene expression levels measured in other cells or body fluids (such as serum, plasma, urine, synovial fluid, or cerebrospinal fluid) in immune system samples taken from individuals without immune system disease Expression levels in tissues or body fluids. Therefore, the present invention provides a diagnostic method for use during the diagnosis of immune system diseases, comprising measuring the gene encoding Neutrokine-α and/or Neutrokine-αSV polypeptide in immune system tissues or other cells or body fluids obtained from an individual. Expression level, and compared with the expression level of standard Neutrokine-α and/or Neutrokine-αSV gene, the expression level of this gene is increased or decreased to indicate immune system disease or normal activation, proliferation, differentiation and/or death.

In particular, it is believed that Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides and proteins encoding Neutrokine-alpha and/or Neutrokine-alphaSV and mRNA are present in certain mammalian tissues with cancer compared to "standard" levels. Expression levels were significantly increased or decreased. In addition, it is believed that in some body fluids (such as serum, plasma, urine, and cerebrospinal fluid) or cells or tissues of such mammals with cancer, the expression levels of Neutrokine-α and/or Neutrokine-αSV polypeptides are comparable to those without cancer. The relative increase or decrease in expression of sera of the same species can be detected.

For example, as described herein, Neutrokine-alpha is highly expressed in cells of the monocytic lineage. Therefore, the polynucleotide of the present invention (such as the polynucleotide sequence complementary to all or part of Neutrokine-α mRNA and/or Neutrokine-αSV mRNA) and the antibody (and antibody fragment) against the polypeptide of the present invention can be used for quantitative or qualitative The concentration of cells of a monocytic lineage (eg, monocytic leukemia cells) expressing Neutrokine-α on their surface. These antibodies are additionally of diagnostic use in detecting abnormal expression levels of the Neutrokine-α gene, or abnormalities in the structure and/or timing, tissue, cellular or subcellular localization of Neutrokine-α and/or Neutrokine-αSV. These diagnostic assays can be performed in vivo or in vitro, for example on blood samples, biopsy tissue or autopsy tissue.

Additionally, as described herein, Neutrokine-alpha receptors are initially expressed on cells of the B cell lineage. Therefore, the Neutrokine-α polypeptide of the present invention (including labeled Neutrokine-α polypeptide and Neutrokine-α fusion protein), and the anti-Neutrokine-α antibody (including fragments of anti-Neutrokine-α antibody) of the polypeptide of the present invention can be used for quantitative Or qualitatively the concentration of B-cell lineage cells (such as B-cell-associated leukemia or lymphoma cells) expressing Neutrokine-α receptors on their surface. These Neutrokine-alpha polypeptides and antibodies are additionally useful in detecting abnormalities in the expression levels of Neutrokine-alpha receptor genes, or in the structure and/or timing, tissue, cellular or subcellular localization of Neutrokine-alpha receptors, and/or in the diagnosis of Neutrokine-alpha receptors -Activity/deficiency in alpha-related signaling pathways has diagnostic use. These diagnostic assays can be performed in vivo or in vitro, for example, on blood samples or biopsies using methods described herein or known in the art.

In one embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention, or the agonist or antagonist of Neutrokine-α and/or Neutrokine-αSV (such as anti-Neutrokine-α and/or anti-Neutrokine-α Neutrokine-αSV) for use in the treatment, prevention, diagnosis or prognosis of individuals suffering from immunodeficiency.

Neutrokine-α and/or Neutrokine-α polynucleotides or polypeptides of the present invention, or Neutrokine-α and/or Neutrokine-αSV agonists or antagonists can be used

(such as anti-Neutrokine-α and/or Neutrokine-αSV antibodies) treatment, prevention, diagnosis and/or prediction of immunodeficiency includes but is not limited to one or more immunodeficiencies selected from the following: X-linked severe combined immunodeficiency ( SCID), autosomal SCID; adenosine deaminase deficiency (ADA deficiency), X-linked agammopathy, (XLA), Bruton's disease, congenital agammopathy, X-linked infantile agammopathy , Acquired Gammaglobulinemia, Adult-Onset Gammaglobulinemia, Late-Onset Gammaglobulinemia, Dysgammaglobulinemia, Hypogammaglobulinemia, Gammaglobulinemia, Common Variability Immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS), X-linked hyper-IgM immunodeficiency, non-X-linked hyper-IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or elevated IgS antibody deficiency, thymoma immunodeficiency, Ig heavy chain deletion, k chain deficiency, B-cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia ( Swiss type), reticulocyte hypoplasia, neonatal neutropenia, severe homogeneous leukopenia, immunodeficiency thymic lymphoid hypoplasia-hypoplasia or dysplasia, ataxia-telangiectasia, short-limbed dwarfism , X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome with combined IgS immunodeficiency, purine nucleotide phosphatase deficiency (PNP), MHC class II deficiency (Bare lymphocytic syndrome), and severe combined immunodeficiency.

According to this embodiment, the level of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV is abnormally low in individuals with immunodeficiency compared to individuals without immunodeficiency. Any method described herein or known in the art can be used to detect Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention (such as FACS analysis or ELISA detection and hybridization or PCR detection), and use For determining the expression pattern of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention in biological samples.

Biological samples from persons with immunodeficiency are characterized by low levels of expression of Neutrokine-α and/or Neutrokine-αSV as compared to that observed in non-immunodeficient individuals. Therefore, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, and/or agonists or antagonists thereof, can be used for diagnosing and/or predicting immunodeficiency according to the methods of the present invention. For example, a biological sample ("target") obtained from a human suspected of having immunodeficiency can be analyzed for relative expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to be immunodeficient. Between the target and control samples, the expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists and/or antagonists thereof are significantly different, suggesting that the target patient have immunodeficiency.

In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention, or an agonist or antagonist thereof (such as an antibody against Neutrokine-α and/or Neutrokine-αSV), is used for Treating, diagnosing and/or prognosing individuals with common variable immunodeficiency disease, (CVID, also known as acquired agammaglobulinemia, and acquired hypogammaglobulinemia) or subclasses thereof. According to this embodiment, individuals with CVID or a subclass thereof exhibit expression levels of Neutrokine-α and/or Neutrokine-αSV receptors on their B cells and/or monocytes when compared to individuals without CVID abnormal. Any method described herein and known in the art can be used to detect Neutrokine-α polynucleotides or polypeptides of the present invention and/or its receptor polypeptides (for example, FACS analysis or ELISA to detect polypeptides, and hybridization to detect polynucleotides) or PCR method), and used to determine the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention and their receptor polypeptides, in samples containing at least monocytes or some components thereof (such as RNA), A different expression pattern than in samples containing at least B cells or components thereof such as RNA. Increased expression of Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide in a sample determined to contain at least monocytes or some component thereof (e.g. RNA), and determined to contain at least B cells or some component thereof (e.g., RNA) samples with Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide expression levels lower than normal, this sample may be suggestive of CVID

(ie acquired agammaglobulinemia, or acquired hypogammaglobulinemia).

Humans with CVID are characterized by elevated expression levels of Neutrokine-alpha and its receptor (NAR) in either peripheral or circulating blood B cells when compared to that observed in humans without CVID. In contrast, individuals without CVID are characterized by low levels of Neutrokine-α expression and high levels of NAR expression in peripheral or circulating blood B cells. Therefore, Neutrokine-α of the present invention, Neutrokine-αSV polypeptide and/or NAR polypeptide, polynucleotide, and/or its agonist or antagonist, can be used in the different diagnosis of this CVID subtype according to the method of the present invention, For example, peripheral blood B cell samples obtained from a person suspected of having CVID (subject) are analyzed for relative expression levels of Neutrokine-α, Neutrokine-αSV and/or NAR polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is compared to the expression level of the same molecule in a human known to be free of CVID (control). Significant differences in the expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, and/or NAR polypeptides of the present invention between the target and control samples suggest that the target suffers from CVID subtype disease.

In a specific embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides, or agonists or antagonists thereof (such as anti-Neutrokine-α and/or anti-Neutrokine-αSV antibodies), are used for diagnosis, prognosis, treatment Or to prevent diseases characterized by defective production of serum immunoglobulins, recurrent infections and/or immune system dysfunction. In addition, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists or antagonists thereof (such as antibodies against Neutrokine-α and/or Neutrokine-αSV), can be used for diagnosis, prediction, treatment or prevention of joint , infection of the bone, skin, and/or parotid gland, blood infection (such as sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune disease (as described herein), inflammation, and malignancy and and/or any disease or dysfunction or pathological condition associated with these diseases, infections, dysfunctions and/or malignancies, including but not limited to CVID, other primary immunodeficiencies, HIV disease, CLL, recurrent bronchitis, Fistula, otitis media, connective tissue inflammation, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe shingles), and/or pneumocystiscapnii.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotide or polypeptide of the present invention or its agonist or antagonist (such as anti-Neutrokine-alpha and/or Neutrokine-alphaSV antibody) is used to treat , to diagnose or predict an individual suffering from an autoimmune disease.

Autoimmune diseases that can be treated, diagnosed or predicted with Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, or agonists or antagonists thereof (such as antibodies against Neutrokine-α and/or Neutrokine-αSV) Including but not limited to one or more of the following diseases: autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, Dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg, IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrine disorders, purpura (eg, Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Pane syndrome, insulin-dependent diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroiditis, Hypothyroidism (ie, Hashimoto's thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, recipient autoimmunity such as (a) Graves' disease, (b) Myasthenia Gravis, and (c) pancreatic Conductin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderm with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, Idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance ( Including treatment of asthma or bladder fibrosis for adrenaline resistance), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland disorders, vitiligo, vasculitis, post-MI, myocardial syndrome, urticaria, genetic atopic dermatitis, asthma, inflammatory myopathy, and other inflammatory, granulamatous, degenerative, and atrophic sexual dysfunctions).

According to this embodiment, an individual with an autoimmune disease or disorder exhibits an abnormally high expression level of Neutrokine-α, Neutrokine-αSV, and/or NAR, as described herein, compared to an individual without an autoimmune disease or disorder. Any method described and known in the art can be used to detect Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, and/or NAR polypeptides (such as FACS analysis or ELISA for detecting polypeptides, and detection of multinuclear Nucleotide hybridization or PCR), and can be used to determine the expression pattern of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or NAR polypeptides in biological samples.

Human biological samples with autoimmune disease are characterized by high expression levels of Neutrokine-α, Neutrokine-αSV and/or NAR when compared to that observed in individuals without autoimmune disease or dysfunction. Therefore, the Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists or antagonists thereof, can be used for diagnosing and/or predicting autoimmune diseases or functions according to the methods of the present invention out of tune. For example, relative expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides and/or NAR polypeptides can be analyzed in a human (target) biological sample obtained from a suspected autoimmune disease or dysfunction. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to be free of an autologous disease. Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides, and/or agonists and/or antagonists thereof, and/or NAR polypeptides of the present invention are expressed between samples from the target and control subjects Significantly different levels suggest that the target body suffers from an autoimmune disease or dysfunction.

In another embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, or agonists or antagonists thereof (such as anti-Neutrokine-α and/or anti-NEUTROKINE-ASV antibodies), are used For the treatment, diagnosis or prognosis of individuals with systemic lupus erythematosus or its subtypes. According to this embodiment, the individual with systemic lupus erythematosus or a subtype thereof expresses abnormally high levels of Neutrokine-α and/or Neutrokine-αSV when compared to individuals without systemic lupus erythematosus. Any method described herein and known in the art can be used to detect Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the present invention (for example, FACS analysis or ELISA to detect polypeptides, and hybridization to detect polynucleotides) or PCR method), and used to determine the expression pattern of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention in biological samples.

Human biological samples with systemic lupus erythematosus are characterized by high expression levels of Neutrokine-α and/or Neutrokine-αSV when compared to that observed in individuals without systemic lupus erythematosus. Therefore, the Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists or antagonists thereof, can be used for diagnosing and/or predicting systemic lupus erythematosus or its relapse according to the present invention. Subtype. For example, biological samples obtained from patients suspected of having systemic lupus erythematosus (object) are analyzed for relative expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention. The expression levels of one or more of these molecules of the invention are then compared to the expression levels of the same molecules in humans known to be free of systemic lupus erythematosus. Significant differences in expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists and/or antagonists thereof, between samples obtained from the target and control subjects, It suggests that the target suffers from systemic lupus erythematosus or its subtypes.

In addition, there is a direct relationship between the severity of systemic lupus erythematosus or its subtypes and the Neutrokine-α and/or Neutrokine-αSV polynucleotide (RNA) and/or polypeptide of the present invention. Therefore, the Neutrokine-α and/or Neutrokine-αSV polynucleotide (RNA), polypeptide and/or agonist or antagonist of the present invention can be used to predict the disease degree of systemic lupus erythematosus or its subtypes according to the method of the present invention. For example, the relative expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention are analyzed in a biological sample obtained from a patient suspected of having systemic lupus erythematosus (target). The expression levels of one or more of these molecules of the invention are then compared to the expression levels of the same molecules in a panel of conditions known to represent varying degrees of the disease. According to this approach, the level of expression corresponds to which member of the group of patients indicates the extent to which that member is present.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotide or polypeptide of the present invention or its agonist or antagonist (such as anti-Neutrokine-alpha and/or Neutrokine-alphaSV antibody) is used to treat , individuals diagnosed or predicted to have rheumatoid arthritis or a subtype thereof. According to this embodiment, the individual with rheumatoid arthritis or a subtype thereof expresses abnormally high levels of Neutrokine-α and/or Neutrokine-αSV compared to individuals without rheumatoid arthritis or a subtype thereof. Any method described herein and known in the art can be used to detect Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention (for example, FACS analysis or ELISA to detect polypeptides, and hybridization to detect polynucleotides) or PCR method), and can be used to determine the expression pattern of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention in biological samples.

Biological samples from patients with rheumatoid arthritis are characterized by increased expression levels of Neutrokine-α and/or Neutrokine-αSV when compared to that observed in individuals without rheumatoid arthritis. Therefore, the Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists or antagonists thereof, can be used for diagnosing and/or predicting rheumatoid arthritis or its subtype. For example, the relative expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention are analyzed in a biological sample obtained from a patient suspected of having rheumatoid arthritis (object). The expression levels of one or more of these molecules of the invention are then compared to the expression levels of the same molecules in humans known to be free of rheumatoid arthritis. The expression levels of Neutrokine-α and/or Neutrokine-αSV polynucleotides and/or polypeptides of the present invention, and/or agonists and/or antagonists thereof are significantly different between samples obtained from the target and control bodies, suggesting Subject has rheumatoid arthritis or a subtype thereof.

Therefore, the present invention provides an effective diagnostic method in diagnosing immune system dysfunction including cancers of this system, and immunodeficiency and/or autoimmune diseases, comprising determining the gene encoding Neutrokine-α and/or Neutrokine-αSV polypeptide, The expression level in the immune system tissue or other cells or body fluids of the individual, and this expression level is compared with the standard Neutrokine-α and/or Neutrokine-αSV gene expression level, so that an increase or decrease compared with the standard level indicates a disease Have an immune system disorder.

Diagnosis of immune system diseases, including but not limited to diagnosis of tumors, immunodeficiency, and/or autoimmune diseases, has been performed according to conventional methods, and the present invention is used as a predictive indicator, so that patients exhibit Neutrokine-α and/or Neutrokine-αSV gene expression levels Enhanced or decreased, relative to patients with gene expression levels near normative levels, exhibited more severe clinical outcomes.

Analyzing or determining the expression level of genes encoding Neutrokine-α and/or Neutrokine-αSV polypeptides refers to directly or indirectly quantitatively or qualitatively measuring or estimating Neutrokine-α and/or Neutrokine-αSV polypeptides or encoding Neutrokine-α and/or The mRNA expression level of Neutrokine-αSV polypeptide in the first biological sample, the direct measurement is to determine or estimate the absolute protein level or mRNA level, and the indirect measurement is to compare with Neutrokine-α and/or Neutrokine-α in the second biological sample

Comparison of SV polypeptide levels or mRNA levels. Preferably, the Neutrokine-α and/or Neutrokine-αSV polypeptide level or mRNA level in the first biological sample is measured or estimated, and compared with the standard Neutrokine-α and/or Neutrokine-αSV polypeptide level or mRNA level, and the standard level is From a second biological sample from a normal human, or by determining the average level in a normal human. Those skilled in the art will appreciate that once a standard Neutrokine-α and/or Neutrokine-αSV polypeptide level or mRNA level is known, it can be repeatedly used as a control standard.

"Biological sample" refers to any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source that contains Neutrokine-α and/or Neutrokine-αSV polypeptide or mRNA. As noted above, biological samples include bodily fluids (such as serum, plasma, urine, synovial fluid, and cerebrospinal fluid) that contain the free extracellular domain of Neutrokine-α and/or Neutrokine-αSV polypeptides, immune system tissues, and others found expressed Organization of intact or free Neutrokine-α and/or the extracellular domain of Neutrokine-αSV or its receptor. Methods for obtaining biopsies and body fluids from mammals are well known in the art. When the biological sample includes mRNA, biopsy tissue is the preferred source.

The compounds of the present invention are useful for diagnosing, prognosing or treating various immune system-related diseases in mammals, preferably humans. Such diseases include, but are not limited to, tumors (eg, B-cell and monocytic leukemias and lymphomas) and tumor metastases, bacterial, viral, and other parasitic infections, immunodeficiency, inflammation, lymphadenopathy, autoimmune diseases (eg, rheumatic arthritis, systemic lupus erythematosus, Sjogren syndrome, mixed connective tissue disease, and inflammatory myopathy), and graft/host disease.

Total cellular RNA can be isolated from biological samples by any suitable method, such as the one-step guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi, Biochemical Analysis 162:156-159 (1987). Levels of mRNA encoding Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides are then analyzed by any suitable method. These methods include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and reverse transcription-ligase chain reaction (RT-LCR).

Biological samples can be assayed for levels of Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides using antibody-based methods. For example, expression of Neutrokine-α and/or Neutrokine-αSV polypeptides in tissues can be studied using traditional immunohistological methods (Jalkanen, M., et al., Journal of Cell Biology 101:976-985 (1985); Jalkanen, M. et al. J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods for detecting Neutrokine-α and/or Neutrokine-αSV polypeptide gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) radioimmunoassay (RIA). Appropriate labels for antibody analysis are known in the art, including enzyme labels such as glucose oxidase, and radioactive isotopes such as iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), Tritium ( ³ H), indium ( ^115m In, ^113m In, ^112m In, ^111m In,), and technetium ( ⁹⁹ Tc, ^99m Tc), titanium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), Molybdenum ( ⁹⁹ Mo) Xenon ( ¹³³ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru; luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin.

Methods known in the art can be used to label the antibodies of the invention. Such methods include, but are not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patent Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; ).

The analyzed tissues or cell types generally include cells or tissues known or suspected to express the Neutrokine-α gene (such as monocyte lineage cells), or cells or tissues known or suspected to express the Neutrokine-α receptor gene (such as B cell lines and splenocytes). Protein isolation methods used in the present invention are, for example, described by Harlow and Lane (Harlow, E and Lane, D., 1988, "Antibody Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), in its entirety incorporated by reference. Isolated cells can be derived from cell culture or from a patient. Analysis of cells taken from culture is a necessary step to determine that the cells can be used as part of a cell-based gene therapy approach, or to test the effect of a compound on Neutrokine-alpha gene or Neutrokine-alpha receptor gene expression.

For example, the antibodies or fragments thereof described herein can be used to quantitatively or qualitatively detect the presence of Neutrokine-alpha gene product or conservative variants or peptide fragments thereof. This can be done, for example, by immunofluorescence methods, detection of fluorescently labeled antibodies by light microscopy, flow cytometry or fluorometry.

Antibodies (or fragments thereof) or Neutrokine-alpha polypeptides of the present invention may additionally be used in histological assays such as immunofluorescence analysis, immunoelectron microscopy or non-immunological assays to detect Neutrokine-alpha gene products or conserved variants thereof in situ. body or peptide fragment, or Neutrokine-α that binds to the Neutrokine-α receptor. In situ detection can be performed by taking a histological sample from a patient, or administering the labeled antibody or Neutrokine-α polypeptide of the present invention thereto. The antibody (or fragment) or Neutrokine-alpha polypeptide is preferably administered by coating the biological sample with a labeled antibody (or fragment). By using this method, not only the Neutrokine-alpha gene product, or conserved variants or peptide fragments, or Neutrokine-alpha polypeptide binding, but also its distribution in the test tissue can be determined. Using the present invention, those skilled in the art will readily appreciate that many histological methods, such as staining methods, can be modified for in situ detection.

Immunological and non-immune assays for the Neutrokine-alpha gene product or its conservative variants or peptide fragments, including in the presence of detectably labeled antibodies that identify the Neutrokine-alpha gene product or its conservative variants or peptide fragments Next, samples such as biological fluids, tissue extracts, freshly harvested cells, or cell lysates incubated in cell culture are incubated, and bound antibody is detected by a number of methods well known in the art.

Immunological and non-immune assays for Neutrokine-alpha receptor gene products or conservative variants or peptide fragments thereof, including in the presence of detectable or Samples such as biological fluids, tissue extracts, freshly harvested cells or cell lysates incubated in cell culture are incubated in the presence of labeled Neutrokine-α polypeptide and bound Neutrokine is detected by a number of methods well known in the art - alpha polypeptide.

The biological sample can be contacted and immobilized on a solid support or carrier, such as nitrocellulose or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support is then washed with an appropriate buffer and subsequently treated with a detectably labeled anti-Neutrokine-alpha antibody or a detectable Neutrokine-alpha polypeptide. The solid support is then washed once more with buffer to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on the solid support can then be detected by conventional methods.

"Solid support or carrier" refers to any support capable of binding antigens or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, ficaite, and magnetite. The nature of the carrier can be soluble in some cases, or insoluble for the purposes of the present invention. The solid support can have virtually any possible configuration, so long as the conjugated molecule is capable of binding the antigen or antibody. Thus, the support configuration may be spherical as in a bead, or cylindrical as in the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface may be flat such as a sheet, test paper, etc. Preferred supports include polystyrene beads. Many other suitable carriers for binding antibodies or antigens are known to those skilled in the art, or can be determined using routine experimentation.

In addition to analyzing Neutrokine-α and/or Neutrokine-αSV polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide levels can also be detected in vivo by imaging . For example, in one embodiment of the invention, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and/or anti-Neutrokine-alpha antibodies are used to visualize B-cell lymphoma. In another embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides and/or anti-Neutrokine-α antibodies and/or Neutrokine-α polynucleotides of the present invention (such as being complementary to all or part of Neutrokine-α and/or or Neutrokine-αSV mRNA) for imaging lymphomas (such as monocytes and B-cell lymphomas).

Antibody labels or markers for in vivo imaging of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides include labels detectable by X-ray, NMR, MRI, CAT scan, or ESR. In the case of x-rays, suitable labels include radioactive isotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the test subject. Appropriate labels for NMR and ESR include labels with a detectable characteristic spin such as tritium, which can be incorporated into antibodies by labeling the relevant hybridoma nutrient. When in vivo imaging is used to detect increased levels of Neutrokine-α and/or Neutrokine-αSV polypeptides for diagnosis in humans, it is preferred to use human antibodies or "humanized" chimeric monoclonal antibodies. Such antibodies can be generated using methods described herein or known in the art. For example, methods for generating chimeric antibodies are known in the art. See, eg, Morrison, Science 229:1202 (1985); Oi et al., Biotechnology 4:214 (1986); Cabilly et al., US Patent 4816567; Taniguchi et al., EP171496; Morrison et al., EP173494; Neuberger et al., WO8601533; WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

Alternatively, any Neutrokine-alpha polypeptide whose presence can be detected can be administered. For example, Neutrokine-alpha polypeptide labeled with a radiopaque or other suitable compound can be administered and the labeled antibody evaluated in vivo as described above. Additionally such Neutrokine-alpha polypeptides may be used in in vitro diagnostic methods.

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide-specific antibodies or antibody fragments, which have been labeled with an appropriate detectable imaging component, are introduced into mammals (e.g., by parenteral, subcutaneous or intraperitoneal administration) to detect immune Systemic diseases, the detectable imaging components are, for example, radioactive isotopes (such as ¹³¹ I, ¹¹² In, ^99m Tc, ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^115m In, ^113m In, ¹¹² In, ¹¹¹ In,), and technetium ( ⁹⁹ Tc, ^99m Tc), titanium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), Palladium ( ¹⁰³ Pd), Molybdenum ( ⁹⁹ Mo) Xenon ( ¹³³ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru), radiopaque material, or material detectable by nuclear magnetic resonance. Those skilled in the art will appreciate that the size of the test subject and the imaging system used will determine the imaging components used to produce the diagnostic image. Where radioactive isotopes are used, the amount of radioactivity injected is typically about 5-20 mCi ^of99mTc in humans. Labeled antibodies and antibody fragments then accumulate preferentially at cells containing the Neutrokine-alpha protein. In vivo tumor imaging is found in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Fragments thereof" (Chapter 13, Imaging Tumors: Radiochemical Detection of Cancer, eds. SW Burchiel and BARhodes, Masson Publishing Inc. (1982)) .

In the case of antibodies, one of the ways in which an anti-Neutrokine-α antibody is detectably labeled is by linking it to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A. "Enzyme-linked immunosorbent Assay (ELISA), 1978, Diagnostic Level 2: 1-7, Microbiology Related Quarter Publications, Walkersville, MD); Voller et al., J. Clin. Pathol. 31: 507-520 (1978); Buter, J.E. Enzymology Methods 73: 482-523 (1981); Maggio, E. et al. (eds.) 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E. et al. (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin , Tokyo). Enzymes with antibody structures will react with appropriate substrates, preferably chromogenic substrates, in such a way as to produce chemical moieties detectable by eg spectrophotometric, fluorometric or visual methods. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate, dehydrogenase, propane Sugar phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6 - Phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Alternatively, detection can be performed by a colorimetric method using a chromogenic substrate reacted with an enzyme. It can also be detected by visual inspection of the reaction of the substrate with the enzyme compared to a similarly prepared standard.

Detection can also be performed by any other immunoassay. For example, Neutrokine-α can be detected by radioimmunoassay (RIA) by radiolabeling antibodies or antibody fragments (see e.g. Weintraub, B., Principles of Radioimmunoassay, 7th training course on radioligand assay methods, The Endocrine Society, March, 1986, incorporated by reference in its entirety). Radioisotopes can be detected by methods including, but not limited to, gamma counters, scintillation counters, or autoradiography.

Antibodies can also be labeled with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence is detectable due to fluorescein. Commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthalaldehyde, and fluorescamine.

Antibodies can also be detectably labeled with fluorescent emitting metals such ^as152Eu , or other lanthanide metals. These metals can be attached to the antibody using chelating groups for the metal, such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Antibodies can also be detectably labeled by coupling them to chemiluminescent compounds. Chemiluminescent-labeled antibodies are then identified by detecting the luminescence that occurs during the chemical reaction. Particularly effective chemiluminescent labeling compounds are eg luminol, isoluminol, theromatic acridinum ester, imidazole, acridinium salt and oxalate.

Additionally, bioluminescent compounds can be used to label the antibodies of the invention. A bioluminescent is a type of chemiluminescence found in biological systems where a catalytic protein increases the potency of the chemiluminescent reaction. The presence of a bioluminescent protein can be detected by detecting the presence of a luminescent substance. Important bioluminescent compounds for labeling include, but are not limited to, luciferin, luciferase, and aequorin.

Treatment of diseases related to the immune system

As mentioned above, Neutrokine-α and/or Neutrokine-αSV polypeptides and polynucleotides and anti-Neutrokine-α antibodies are used to diagnose diseases with abnormally high or low expression of Neutrokine-α and/or Neutrokine-αSV activity. Cells and tissues wherein Neutrokine-alpha and/or Neutrokine-alphaSV are expressed and whose activity is regulated by Neutrokine-alpha and/or Neutrokine-alphaSV, Neutrokine-alpha and/or Altered (increased or decreased) expression levels of Neutrokine-αSV produce pathological changes associated with the body system in which Neutrokine-α and/or Neutrokine-αSV is expressed and/or active.

Those skilled in the art also know that since the Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention are members of the TNF family, the extracellular domain of the representative protein can be expressed in a soluble form from Neutrokine-α and/or Neutrokine-αSV released by proteolysis, and therefore when Neutrokine-α and/or Neutrokine-αSV polypeptides [especially soluble forms of representative extracellular domains] are added from exogenous sources to an individual's tissue, cell or body, the The polypeptide exhibits a modulated activity on any of its target cells in an individual. Likewise, cells expressing this type II transmembrane protein can be added to individual cells, tissues or organisms, whereby the added cells bind to cells expressing Neutrokine-α and/or Neutrokine-αSV receptors, thereby expressing Neutrokine-α and/or Neutrokine-αSV can have an effect (such as proliferation or cytotoxicity) on target cells bearing the receptor.

In one embodiment, the present invention provides a composition comprising a polypeptide of the present invention (such as containing Neutrokine-α and/or Neutrokine-αSV associated with a heterologous polypeptide, heterologous nucleic acid, toxin or prodrug) for targeted cell delivery. Polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-αSV antibody composition), the targeted cell is a B cell expressing Neutrokine-α and/or Neutrokine-αSV receptor, or expressing a cell surface bound form Neutrokine-α and/or Neutrokine-αSV monocytes. Neutrokine-α and/or Neutrokine-αSV polypeptides or anti-Neutrokine-α and/or Neutrokine-αSV antibodies of the present invention interact with heterologous polypeptides, heterologous nucleic acid, toxin or prodrug combination.

In one embodiment, the present invention provides a method for specifically delivering a composition of the present invention to a cell by administering a polypeptide of the present invention (such as Neutrokine-α and/or Neutrokine-alpha) associated with a heterologous polypeptide or nucleic acid. αSV polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-α antibody). In one embodiment, the invention provides a method of delivering a therapeutic protein to a targeted cell. In another embodiment, the present invention provides a single-stranded nucleic acid (such as an antisense or ribozyme molecule), or a double-stranded nucleic acid (such as DNA that can be integrated into the genome of a cell or additionally replicated and transcribed) .

In another embodiment, the present invention provides a method for specifically destroying cells (such as destroying tumor cells), by administering a polypeptide of the present invention (such as Neutrokine-α and/or Neutrokine-αSV polypeptide or anti-Neutrokine-α and/or anti-Neutrokine-αSV antibody).

In a specific embodiment, the present invention provides a method for specifically destroying cells of the B cell lineage (such as B cell-associated leukemia or lymphoma) by administering Neutrokine-α in combination with a toxin or a cytotoxic prodrug and/or Neutrokine-αSV polypeptide.

In another specific embodiment, the present invention provides a method of specifically destroying cells of monocytic lineage (such as monocytic leukemia or lymphoma) by administering an anti-Neutrokine associated with a toxin or a cytotoxic prodrug. -α and/or anti-Neutrokine-αSV antibody.

"Toxin" means a compound that binds to and activates an endogenous cytotoxic effector system, a radioisotope, a holotoxin, a modified toxin, a catalytic subunit of a toxin, a cytotoxin (cytotoxic agent), or a cell death-causing Any molecule or enzyme that is not normally present on or on the surface of a cell under conditions. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioactive isotopes, compounds known in the art such as antibodies (or complement fixation regions comprising a portion thereof) that bind intrinsic or induced endogenous cytotoxic effector systems, Thymidine kinase, endonuclease, RNase, α-toxin, ricin, red bean toxin, Pseudomonas exotoxin A, diphtheria toxin, saporin, charantin, gelonin, pokeweed antiviral protein, α-sarcin and cholera toxin. "Toxin" also includes cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, such as alpha-emitters such as ²¹³ Bi, or other radioactive isotopes such as ¹³³ Xe, ¹³¹ I, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ Yttrium, ¹¹⁷ Tin, ¹⁸⁶ Rhenium, ¹⁶⁶ Holmium and ¹⁸⁸ Rhenium, luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

Methods known in the art can be used to label the antibodies of the invention. Such methods include, but are not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patents 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; ). Cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thiophene glucoside, vincristine, vinca Alkaline, colchicine, doxorubicin, daunorubicin, dione mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, Tetracaine, lidocaine, propranolol, and puromycin and their analogs or congeners. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, dichloromethyldiethylamine, thioepa Chlorambucil, phenylalanine mustard, carmustine (BSNU) and cyclohexylnitrosourea (CCNU), cyclothosphamide, busulfan, bromomannitol, streptozotocin, mitomycin C, and dichlorodiamine cisplatin(II) (DDP), anthracyclines (such as daunorubicin, (formerly daunomycin) and doxorubicin), antibiotics (such as daunorubicin ( formerly actinomycin), bleomycin, mithramycin, anthranimycin (AMC)), and antimitotic agents (eg, vincristine and vinblastine).

"Cytotoxic prodrug" refers to a non-toxic compound that is converted to a cytotoxic compound by enzymes normally present in the cell. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include, but are not limited to, glutamyl derivatives of benzoic acid sinapyl alkylating agents, epipodophyllotoxin glucopyranoside or phosphate derivatives of mitomycin C, Cytarabine, daunorubicin and phenoxyacetamide derivatives of doxorubicin.

It should be known that diseases caused by Neutrokine-α and/or Neutrokine-αSV activity levels below standard or normal levels in individuals, especially immune system diseases, can be treated by administering Neutrokine-α and/or Neutrokine-αSV polypeptides (in the form of soluble extracellular domain or cellular form expressing the intact protein) or agonists. Accordingly, the present invention also provides a method of treating an individual in need of increased levels of Neutrokine-α and/or Neutrokine-αSV activity, comprising administering to the individual a pharmaceutical composition comprising an amount of the isolated Inventing Neutrokine-α and/or Neutrokine-αSV polypeptides or agonists thereof effectively increases the level of Neutrokine-α and/or Neutrokine-αSV activity in such individuals.

It should also be known that diseases caused by individual Neutrokine-α and/or Neutrokine-αSV activity levels higher than standard or normal levels, especially immune system diseases, can be treated by administering Neutrokine-α and/or Neutrokine-αSV polypeptide (soluble cell ectodomain or a cellular form expressing the intact protein) or its antagonist (such as an antibody against Neutrokine-α). The present invention therefore also provides a method of treating an individual in need of reduced Neutrokine-α and/or Neutrokine-αSV activity, comprising administering to such an individual a pharmaceutical composition comprising an amount of the isolated Neutrokine of the present invention - an alpha and/or Neutrokine-alphaSV polypeptide, or an antagonist thereof, effective to reduce the level of Neutrokine-alpha and/or Neutrokine-alphaSV activity in such an individual.

The Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides or their antagonists of the present invention can be used to treat infectious diseases. For example, infectious diseases can be treated by increasing the immune response, particularly B cell proliferation and differentiation. The immune response can be enhanced by enhancing an existing immune response, or by initiating a new immune response. Alternatively, Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polynucleotides or polypeptides, or antagonists thereof, can also directly inhibit infectious agents without eliciting an immune response.

A virus is, for example, an infectious agent that produces a disease or clinical condition that can be treated by Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide or an agonist thereof. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and virus families: Arboviruses, Adenoviruses, Arenaviruses, Arteriviruses, DiRNAviruses, Buniaviruses, Caliciviruses, Circoviridae, Coronaviruses, Dengue Viruses, EBV, HIV, flavivirus, hepadnavirus (hepatitis virus), herpesvirus (eg, cytomegalovirus, herpes simplex virus, herpes zoster virus), mononegavirus (eg, paramyxovirus, measles virus, rhabdovirus) , orthomyxoviruses (eg, influenza A, B, and parainfluenza), papillomaviruses, papovaviruses, parvoviruses, picornaviruses, poxviruses (eg, smallpox or vaccinia), reoviruses (such as rotavirus), retroviruses (HTLV-I, HTLV-II, lentiviruses), and togaviruses (such as rubella virus). Viruses in these families can cause a variety of diseases or clinical symptoms, including but not limited to arthritis, bronchitis, respiratory syncytial virus, encephalitis, ocular infections (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Type 6), Japanese Encephalitis B, Junin, Chikungunya, Rift Valley Fever, Yellow Fever, Meningitis, Opportunistic Infections (eg AIDS), Pneumonia, Burkitt's Lymphatic tumors, varicella, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold poliomyelitis, leukemia, rubella, venereal diseases, skin diseases (such as Kaposi' warts) and viremia. Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists or antagonists thereof, can be used in the treatment, prevention, diagnosis and/or detection of any of these symptoms or diseases. In specific embodiments, Neutrokine-alpha-polynucleotides, polypeptides or agonists are used in the treatment, prevention and/or diagnosis of: meningitis, dengue fever, EBV, and/or hepatitis (eg B). In another specific embodiment, Neutrokine-[alpha]-polynucleotides, polypeptides or agonists are used to treat patients who are unresponsive to one or more other commercially available hepatitis vaccines. In another specific embodiment, Neutrokine-alpha polynucleotides, polypeptides or agonists are used in the treatment, prevention and/or diagnosis of AIDS. In another specific embodiment, Neutrokine-α and/or Neutrokine-αSV and/or its receptor polynucleotides, polypeptides, agonists and/or antagonists are used for the treatment, prevention and/or diagnosis of Cryptosporidiosis .

Similarly, bacterial or fungal etiologies that can cause disease or clinical symptoms and are treatable with Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, include, but are not limited to, the following Gram-negative and Gram-negative Lambert-positive bacteria and bacterial families and fungi: Actinomycetes (such as Corynebacterium, Mycobacterium, Nocardia), Cryptococcus neoformans, Aspergillus, Bacillus family (such as Anthracis, Clostridium), Bacteroides, Blastomycetes, Bordetella, Borrelia (eg, Borrelia Burgdorferi), Brucella, Candida, Campylobacter, Coccidioides, Cryptococci, Dermatocycoses, Escherichia coli (eg, enterotoxigenic Escherichia coli coli and enterohaemorrhagic Escherichia coli), Enterobacter species (Klebsiella, Salmonella (e.g. Salmonella typhi, Paratyphi), Serratiia, Yersinia), Erysipelothrix, Helicobacter, Legionella Genus, Leptospira, Listeria (eg, Listeria monocytogenes), Mycoplasma, Bacillus leprae, Vibrio cholerae, Neisseria (eg, Actinobacillus, Gornorrhea, meningococcus), Meisseriameningitidis, Pasteurella infection (eg, Actinobacillus, Haemophilus (eg, Haemophilus influenzae type B), Pasteurella), Pseudomonas, Rickettsia, Chlamydia, Syphilis, Shigella, Staphylococcus , meningococci, pneumococci, and streptococci (such as Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can produce diseases or symptoms including, but not limited to, bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (such as AIDS-related infections), paronychia, prosthesis-related infections, Reiter's disease, respiratory infections such as whooping cough or empyema, sepsis, Lyme disease, Cat-Seratch disease, dysentery, paratyphoid, food poisoning, typhoid, pneumonia, gonorrhea, meninges Inflammation (such as meningitis A, B), chlamydial disease, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, venereal disease, skin disease (such as cellulitis, dermatocycoses), toxaemia, urinary tract infection, wound infection. Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, are useful in the treatment, prevention, diagnosis and/or detection of any of these diseases or conditions. In specific embodiments, Neutrokine-alpha polynucleotides, polypeptides or agonists thereof are used in the treatment, prevention and/or diagnosis of: Tetanus, Diptheria, Botulism and/or Meningitis B.

In addition, parasitic etiologies that cause disease or symptoms and that can be treated with Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists thereof, include but are not limited to the following families or types: amoebiasis, amoebiasis Besidiosiosis, Coccidioides, Cryptosporidiosis, Dientamoebiasis, Phytophthora, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Tyler's Pyroplasmosis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and sporozoites (eg, Plasmodium virax, Plasmodium falciparum, Plasmodium malaria, and Plasmodium ovale). These parasites can cause a variety of diseases or conditions, including but not limited to scabies, fall scrub typhus, eye infections, intestinal disorders (eg, dysentery, giardiasis), liver disease, lung disease, opportunistic infections (eg, AIDS-related infections), malaria, pregnancy complications, and toxoplasmosis. Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists or antagonists thereof, may be used in the treatment, prevention, diagnosis and/or detection of any of these symptoms or diseases. In specific embodiments, Neutrokine-alpha-polynucleotides, polypeptides or agonists thereof are used in the treatment, prevention and/or diagnosis of malaria.

In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention are used for the treatment, prevention and/or diagnosis of inner ear infections (such as otitis media ), and other infections characterized by Streptococcus pneumoniae and other pathogenic microorganisms.

In a specific embodiment, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or polypeptides, or agonists or antagonists thereof (such as antibodies against Neutrokine-alpha and/or Neutrokine-alphaSV), are used to treat or Prevention of diseases characterized by defective production of serum immunoglobulins, recurrent infections and/or immune system dysfunction. In addition Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists or antagonists thereof (such as anti-Neutrokine-α and/or Neutrokine-αSV antibodies), can be used to treat or prevent joints, bones, skin and and/or parotid gland infection, blood-induced infection (such as sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune system disease (as described herein), inflammation, and malignancy, and/or with Any disease or dysfunction or pathological change associated with these infections, diseases, dysfunctions, or malignancies, including but not limited to CVID, other primary immunodeficiencies, HIV, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis , pneumonia, hepatitis, meningitis, herpes zoster (eg, severe shingles), and/or Pneumocystis carinii.

Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists or antagonists thereof, may be used in the diagnosis, prediction, treatment or prevention of one or more of the following diseases or disorders, or pathological conditions: primary immunodeficiency , immune-mediated thrombocytotic anemia, Kawasaki syndrome, bone marrow transplantation (eg, in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (eg, in adults or children with HIV infection), chronic inflammatory demyelination, and more Neuropathy, and post-transfusion purpura.

In addition, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, or agonists or antagonists thereof, can be used to diagnose, predict, treat or prevent one or more of the following diseases, dysfunctions or pathological conditions: Guillian-Barre syndrome, anemia (eg, parvovirus B19-associated anemia, patients with stable multiple myeloma who are at high risk for infection (eg, recurrent infection), autoimmune hemolytic anemia (eg, febrile autoimmune hemolytic anemia), thrombocytotic anemia (eg, neonatal thrombocytosis), and immune-mediated neurogenic anemia), transplantation (eg, CMV-negative recipient of a CMV-positive organ), hypogammaglobulinemia ( (eg, neonatal hypogammaglobulinemia with risk factors for infection or morbidity), epilepsy (eg, refractory epilepsy), systemic vascular syndrome, myasthenia (eg, cardiac decompensation in myasthenia), dermatomyositis , and polymyositis.

Additional preferred embodiments of the present invention include, but are not limited to, using Neutrokine-α and/or Neutrokine-αSV polypeptides, polynucleotides and functional agonists thereof for the following applications:

Administration to animals (such as mice, rats, rabbits, hamsters, guinea pigs, pigs, piglets, chickens, camels, goats, horses, cattle, sheep, dogs, cats, non-human primates, and humans, preferably humans), with Assisting the immune system to produce increased amounts of one or more antibodies (such as IgG, IgA, IgM, and IgE), induce higher affinity antibody production (such as IgG, IgA, IgM, and IgE), and/or enhance the immune response. In a specific embodiment, the Neutrokine-alpha polypeptides of the invention and/or agonists thereof are administered to assist the immune system in producing increased amounts of IgG. In another specific embodiment, the Neutrokine-alpha polypeptides of the invention and/or agonists thereof are administered to assist the immune system in producing increased amounts of IgA. In another specific embodiment, the Neutrokine-alpha polypeptides of the invention and/or agonists thereof are administered to assist the immune system in producing increased amounts of IgM.

Administration to animals (including but not limited to those listed above and also including transgenic animals) that are unable to produce functional endogenous antibody molecules, or that have an otherwise composed endogenous immune system, but cannot be obtained by reconstitution from another animal or Human immunoglobulin molecules are produced by partially reconstituting the immune system (see, eg, PCT Publication Nos. WO98/24893, WO96/34096, WO96/33735 and WO91/10741).

A vaccine adjuvant that enhances the immune response to a specific antigen. In a specific embodiment, the vaccine adjuvant is Neutrokine-α and/or Neutrokine-αSV polypeptides as described herein. In another specific embodiment, the vaccine adjuvant is Neutrokine-α and/or αSV polynucleotide as described herein (i.e. Neutrokine-α and/or Neutrokine-αSV polynucleotide is a genetic vaccine adjuvant ). As described herein, Neutrokine-α and/or Neutrokine-αSV polynucleotides of the invention can be administered using methods known in the art, including but not limited to liposome delivery, recombinant vector delivery, injection of naked DNA, and gene gun delivery.

An adjuvant enhances tumor-specific immune responses.

An adjuvant enhances the antiviral immune response. The antiviral immune response can be enhanced by using the composition of the present invention as an adjuvant, and the antiviral immune response includes, but is not limited to, viruses and virus-related diseases or symptoms described in the present invention or known in the art. In specific embodiments, the compositions of the invention are used as adjuvants to enhance the immune response to a viral disease or condition selected from AIDS, meningitis, dengue, EBV, and hepatitis (eg, hepatitis B). In another specific embodiment, the composition of the present invention is used as an adjuvant to enhance the immune response to a virus, disease or condition selected from the group consisting of: HIV/AIDS, Respiratory Syncytial Virus, Dengue, Rotavirus, Japanese Encephalitis B, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley Fever, Herpes Simplex, and Yellow Fever. In another specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response to the HIV gp120 antigen.

An adjuvant that enhances the antibacterial or antifungal immune response. Antibacterial or antifungal immune responses enhanced by the compositions of the present invention as adjuvants include bacteria or fungi and diseases or conditions associated with bacteria or fungi described herein and known in the art. In specific embodiments, the compositions of the invention are used as adjuvants to enhance the immune response to bacteria or fungi, diseases or conditions selected from the group consisting of tetanus, diphtheria, botulism, and B meningitis. In another specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response to bacteria or fungi, diseases or conditions selected from: Vibrio cholerae, Bacillus leprae, Bacillus typhi, Bacillus paratyphi , Meisseria meningitidis, Streptococcus pneumoniae, group B Streptococcus, Shigella, enterotoxigenic E. coli, enterohaemorrhagic E. coli, Borreliaburgdorferi, Plasmodium spp. (malaria).

An adjuvant enhances the antiparasitic immune response. The immune response against parasites, including parasites and parasite-associated diseases or conditions described herein or known in the art, can be enhanced using the compositions of the present invention as adjuvants. In a specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response to parasites. In another specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response against Plasmodium (malaria).

Acts as a stimulator of B cell responses to pathogens.

As a factor to evaluate the individual's immune status before receiving immunosuppressive therapy.

As a factor that induces high-affinity antibodies.

Acts as a factor that increases serum immunoglobulin concentrations.

Factors that act as accelerated immunity contain individual recovery.

As a factor that assists the immune response in the elderly.

As an enhancer of the immune system before, during or after bone marrow transplantation and/or other transplantation such as allogeneic or xenogeneic organ transplantation. With regard to transplantation, the compositions of the present invention may be administered prior to, simultaneously with, and/or after transplantation. In a specific embodiment, the composition of the invention is administered after transplantation but before T cell recovery begins. In another specific embodiment, the composition of the invention is first administered after initial restoration of T cell populations following transplantation, but before complete restoration of B cells.

As a factor in the immune response in individuals with helper B-cell immunodeficiency, such as patients after partial or complete splenectomy. B cell immunodeficiencies that can be improved or treated by administering Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, include but are not limited to X-linked severe combined immunodeficiency (SCID) , autosomal SCID, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, agammaglobulinemia, hypogammaglobulinemia, infant transient hypogammaglobulinemia, nonspecific Heterosexual hypogammaglobulinemia, agammaglobulinemia, common variant immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS), X-linked hyper IgM immunodeficiency, non-X-linked hyper IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or high IgS antibody deficiency, thymoma immunodeficiency, Ig heavy chain deletion, kappa chain deficiency, B-cell lymphoproliferative disorder (BLPD ), selective IgM immunodeficiency, recessive gammopathy (Swiss type), reticular hypoplasia, neonatal neutropenia, severe congenital leukemia, immunodeficiency thymic lymphoid hypoplasia, exercise Dysregulated telangiectasia, short-limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nozelof syndrome combined with Igs immunodeficiency, purine nucleoside phosphorylase (PNP) deficiency, MHC class II deficiency (Bare lymphocyte syndrome), and severe combined immunodeficiency.

As a preparation for boosting the immune response of individuals with acquired loss of B cell function, acquired B cells that can be improved or treated by administering Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof Loss of function diseases include, but are not limited to, HIV infection, AIDS, bone marrow transplantation, and B-cell chronic lymphocytic leukemia (CLL).

Acts as a factor that assists the immune response in individuals with transient immunodeficiency. Diseases that can be improved or treated by administering Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, including but not limited to viral infections (such as influenza), and malnutrition Associated illness, mononucleosis infection, or stress-related illness, measles, blood transfusion, surgery.

Acts as a modulator of antigen presentation by monocytes, dendritic cells and/or B cells. In one embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides (soluble, membrane-bound or transmembrane forms) or polynucleotides enhance antigen presentation or antagonize antigen presentation in vitro or in vivo. Additionally, in related embodiments, such enhancement or antagonism of antigen presentation may be used in anti-tumor therapy or modulation of the immune system.

Acts as a mediator of the mucosal immune response. Expression of Neutrokine-α by monocytes and other responses by B cells suggests that it may be involved in the exchange of signals between B cells and monocytes or between their differentiated progeny. This activity is in many ways identical to CD40-CD154 signaling between B cells and T cells. Therefore, Neutrokine-α may be an important regulator of T cell-independent immune responses to environmental pathogens. The unconventional B cell population (CD5+), especially associated with the mucosa and responding to many innate immune responses in the human body, responds to Neutrokine-alpha, thus enhancing the protective immune status of the individual.

As a factor that directs the individual's immune system to produce a humoral response (ie TH2) that is opposite to the TH1 cell response.

Acts as a factor that induces tumor proliferation and thus makes it more suitable for antineoplastic agents. For example, multiple myeloma is a slowly differentiating disease and is therefore refractory to virtually all antineoplastic approaches. If these cells were made to proliferate faster, their receptivity to anti-tumor approaches would be improved.

As a B cell specific binding protein, specific activators or inhibitors of cell growth can be attached to it. The result is to focus the activity of this activator or inhibitor on normal, diseased or neoplastic B cell populations.

As a factor to detect B cell lineage cells by their specificity. This application may require labeling the protein with biotin or other agents (as described herein) for detection.

As a stimulator of B cell production in diseases such as AIDS, chronic lymphocyte dysfunction and/or general variable immunodeficiency.

It functions to isolate B cells from a heterogeneous mixture of cell types as part of a method for selecting B cells. Neutrokine-α can be coupled with a solid support, and then B cells can specifically bind to it. Unbound cells are washed away and bound cells are subsequently eluted. One non-limiting application of this option is to deplete tumor cells from, for example, bone marrow or peripheral blood prior to transplantation.

As a therapy to generate and/or regenerate lymphoid tissue following surgery, trauma or genetic defect.

Use as a gene therapy for genetic diseases that result in incompatibility as observed in SCID patients.

Serves as an antigen that suppresses or enhances Neutrokine-α-mediated responses.

As a substance to activate monocytes/macrophages against parasitic diseases that act on monocytes, such as leishmaniasis.

As a pretreatment agent for bone marrow samples prior to transplantation. This treatment increases B cell re-presentation and thus accelerates recycling.

As a substance that regulates secreted cytokines, the secretion of which is stimulated by Neutrokine-α.

Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, can be used to modulate IgE concentrations in vitro or in vivo.

In addition, the Neutrokine-α or Neutrokine-αSV polypeptides or nucleotides of the present invention, or agonists thereof, can be used in the treatment, prevention and/or diagnosis of IgE-mediated allergy. Such allergies include, but are not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat, prevent, diagnose and/or ameliorate selective IgA deficiency.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide of the present invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate ataxia telangiectasia .

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, diagnose and/or ameliorate common variant immunodeficiency.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide of the present invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate X-linked gamma globulin deficiency disease.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide of the present invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate severe combined immunodeficiency (SCID) .

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, diagnose and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, diagnose and/or ameliorate X-linked hyper IgM Ig defect.

In another specific embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or agonists or antagonists thereof (such as anti-Neutrokine-α antibodies), are administered to treat, prevent, diagnose and/or diagnose chronic myelogenous leukemia, acute myelogenous leukemia, leukemia, hystiocytic leukemia, monocytic leukemia (such as acute monocytic leukemia), reticulocytic leukemia, Shilling type monocytic leukemia, and /or other leukemias derived from monocytes and/or mononuclear cells and/or tissues.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide of the present invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate monocytic leukemia , for example in tuberculosis.

In another specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polypeptide or polynucleotide of the present invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate, monocytic leukocytosis syndrome, monocytic leukopenia, monocytic anemia, and/or mononucleosis.

In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention, and/or anti-Neutrokine-α antibodies and/or agonists or antagonists thereof, are used for treatment, prevention , detecting and/or diagnosing primary B lymphocyte dysfunction and/or diseases and/or pathological conditions associated therewith. In one embodiment, such primary B lymphocyte dysfunction, disease and/or pathological condition is characterized by complete or partial loss of humoral immunity. Primary B lymphocyte dysfunction, disease and/or pathological condition characterized by complete or partial loss of humoral immunity, which can be prevented, treated, detected and/or diagnosed with the composition of the present invention, including but not limited to X-linked blood Moderate gammaglobulin deficiency (XLA), severe combined immunodeficiency (SCID), and selective IgA deficiency.

In a preferred embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides, polypeptides and/or agonists or antagonists thereof are used for treatment, prevention and/or diagnosis of one or more various Mucosa-related diseases. Such diseases include, but are not limited to, mucositis, mucosectomy, mucinous colitis, mucosal and cutaneous leishmaniasis (e.g., American leishmaniasis, nasopharyngeal leishmaniasis, and New World leishmaniasis ), mucocutaneous lymphoid syndrome (eg, Kawasaki disease), mucinous enteritis, mucinous epidermoid carcinoma, mucinous epidermoid neoplasm, mucoepithelial dysplasia, mucinous adenocarcinoma, mucinous degeneration, myxoid degeneration; myxoma Degeneration; myxomatosis multiplex, myxoid medial hypertrophy [eg, medial necrosis], mucolipidosis (including type I, type II, type III, and type IV), Mucoid enteritis, mucopolysaccharidosis (eg, mucopolysaccharidosis type I (ie, Hurler's syndrome), mucopolysaccharidosis type IS (ie, Scheie's syndrome or mucopolysaccharidosis type V), mucopolysaccharidosis type II (ie, Hunter's syndrome) , type III mucopolysaccharidosis (ie Sanfilippo's syndrome), type IV mucopolysaccharidosis (ie Morquio's syndrome), type VI mucopolysaccharidosis (ie Naroteaux-Lamy syndrome), type VII mucopolysaccharidosis (ie due to β-gluco Mucopolysaccharidosis due to uronidase deficiency), and (mucosulfatosis) mucopolysaccharidosis, mucopurulent conjunctivitis, mucopurulent, mucormycosis, mucosal disease (ie, bovine viral diarrhoea), mucinous colitis (such as mucous and mucous membraneous colitis), and pancreatic fibrocystic disease (such as pancreatic fibrosis, Carke-Hadfield syndrome, pancreatic fibrocystic disease, pancreatic fibrocystic disease). In a particularly preferred In an embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides, polypeptides and/or agonists and/or antagonists thereof are used for the treatment, prevention and/or diagnosis of mucositis, especially mucositis associated with chemotherapy.

In a preferred embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides, polypeptides and/or agonists and/or antagonists thereof are used for the treatment, prevention and/or diagnosis of diseases associated with sinusitis.

Another disease or condition for which Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists thereof, can be treated, prevented and/or diagnosed is osteomyelitis.

Another disease or condition for which Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or agonists thereof, may be treated, prevented and/or diagnosed is endocarditis.

All of the above applications can be used in veterinary pharmacy.

Antagonists of Neutrokine-alpha include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes, and Neutrokine-alpha polypeptides of the invention. These can be expected to reverse some of the activities of the aforementioned ligands, as well as to find some clinical or practical applications, such as:

Used to block all aspects of the immune response to foreign or self substances. Examples include autoimmune diseases such as lupus, and arthritis, as well as immune responses to skin allergies, inflammation, intestinal disease, injury, and pathogens. Although the available data directly illustrate the potential role of Neutrokine-α in B-cell and monocyte-associated pathology, other cell types may also express or respond to Neutrokine-α. Therefore, Neutrokine-α, like CD40 and its ligands, can be regulated by the status of the immune system and the microenvironment of the cells.

In the treatment of preventing B cell proliferation and Ig secretion associated with autoimmune diseases, such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

As an inhibitor of graft versus host disease or transplant rejection.

Treatment of B cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma, and EBV-transformed disease.

Treatment of chronic hypergammaglobulinemia, such as in unicellular gammopathy of undetermined significance (MGUS), Waldenstrom's disease, relatively idiopathic unicellular gammopathy, and plasmacytoma.

Treatment reduces cell proliferation in large B-cell lymphoma.

Decreases B cell and Ig correlators associated with chronic myelogenous leukemia.

as an immunosuppressant.

Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or antagonists thereof, can be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of Neutrokine-α and/or Neutrokine-αSV polypeptides or polynucleotides of the present invention, or antagonists thereof, may be used in the treatment, prevention and/or diagnosis of IgE-mediated allergy, including but not Limited to asthma, rhinitis and eczema.

Acts as an inhibitor of a signaling pathway involving ERK1, COX2, and Cyclin D2, which is associated with Neutrokine-α-induced B cell activation.

The above applications are available in a wide variety of hosts. Such hosts include, but are not limited to, humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, piglets, chickens, cows, sheep, dogs, cats, non-human primates, and people. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In a more preferred embodiment, the host is human.

Agonists and antagonists can be used in combination with pharmaceutically suitable carriers.

Antagonists are useful, for example, to inhibit Neutrokine-α-mediated and/or Neutrokine-αSV-mediated chemotaxis and activation of macrophages and their precursors, and neutrophils, basophils, B lymphocytes, and Certain T cell subsets such as activated and CD8 cytotoxic T cells, and natural killer cells, are involved in some autoimmune diseases and chronic inflammatory and infectious diseases. Autoimmune diseases such as multiple sclerosis, and insulin-dependent diabetes mellitus, antagonists are also useful in the treatment, prevention and/or diagnosis of infectious diseases, including silicosis, sarcoidosis, primary pulmonary fibrosis, by preventing Nuclear phagocyte recruitment and activation. They are also useful in the treatment, prevention and/or diagnosis of primary eosinophilic syndrome by preventing eosinophil production and migration. Endotoxic shock can also be treated by this antagonist, by preventing macrophage migration and their production of Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention. This antagonist is also useful in the treatment of arteriosclerosis by preventing monocytes from infiltrating the arterial wall. This antagonist can also be used in the treatment, prevention and/or diagnosis of histamine-mediated allergy and immune dysfunction, including late allergy, chronic urticaria, and atopic dermatitis, by inhibiting chemokine-induced mast cells and Degranulation of basophils and release of histamine. It can also treat IgE-mediated allergies such as allergic asthma, rhinitis and eczema. Antagonists are also useful in the treatment, prevention and/or diagnosis of chronic and acute inflammation by preventing the invasion of monocytes into the damaged area. They can also be used to modulate normal lung macrophage populations, as chronic and acute lung inflammation is associated with the recruitment of monocyte-macrophages within the lung. Antagonists are also useful in the treatment, prevention and/or diagnosis of rheumatoid arthritis by preventing the invasion of monocytes into the synovial fluid in the patient's joints. Monocyte influx and activation play an important role in degenerative and inflammatory arthropathies. Antagonists can be used to interfere with the harmful linkages to the body caused by IL-1 and TNF by preventing the biosynthesis of other inflammatory cytokines. In this manner, antagonists can be used to prevent inflammation. Antagonists are also useful for inhibiting prostaglandin-independent fever induced by Neutrokine-α and/or Neutrokine-αSV. Antagonists are also useful in the treatment, prevention and/or diagnosis of myeloid disorders such as aplastic anemia and myelodysplastic syndrome. Antagonists are also useful in the treatment, prevention and/or diagnosis of asthma and allergy by preventing the accumulation of eosinophils in the lungs. Antagonists are also useful in the treatment, prevention and/or diagnosis of subepithelial basement membrane fibrosis, a distinct feature of asthmatic lungs. Antagonists are also useful in the treatment, prevention and/or diagnosis of lymphomas (such as, but not limited to, one or more of the lymphomas listed herein).

All of the above applications can be used in veterinary pharmacy.

Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention can be used for treatment, prevention, and/or diagnosis of various immune system-related diseases in mammals, preferably humans . Many autoimmune diseases result from inappropriate recognition of self as foreign by immune cells. This inappropriate recognition results in an immune response that destroys host tissues. Thus, administration of the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention can inhibit the immune response, particularly the proliferation of B cells and/or the production of immunoglobulins , can effectively treat and/or prevent autoimmune diseases. Therefore, in a preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV antagonists of the present invention (such as Neutrokine-α and/or Neutrokine-αSV polypeptide fragments and anti-Neutrokine-α antibodies) are used for treatment, prevention and/or diagnose an autoimmune disease.

Autoimmune diseases that can be treated, prevented and/or diagnosed with Neutrokine-α polynucleotides of the present invention, polypeptides and/or antagonists (such as anti-Neutrokine-α antibodies), including but not limited to autoimmune hemolytic anemia, autoimmune Neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cell reduction, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, nephropathy Glomeronephritis (ie, IgA nephropathy), multiple sclerosis, neuritis, uveitis, polyendocrinopathy, purpura (eg, Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune ocular inflammation.

Additional autoimmune diseases that may be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, autoimmune thyroiditis, subthyroiditis (i.e., Hashimoto's thyroiditis), (usually characterized by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erythematosus (usually characterized by circulating blood and local production of immune complexes), Goodpasture's syndrome (usually characterized by anti-basement membrane antibodies), recipient autoimmunity such as (a) Grave's disease (usually characterized by TSH receptor antibodies), (b) Myasthenia Gravis (usually characterized by acetylcholine receptor antibodies) and (c) insulin resistance (usually characterized by insulin receptor antibodies), autoimmune hemolytic anemia (usually characterized by Phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (usually characterized by phagocytosis of antibody-sensitized platelets).

Additional autoimmune diseases that may be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, rheumatoid arthritis (usually characterized by immune complex characterization in the joints), schleroderm with anti-collagen antibodies (usually characterized by in nucleolar antibodies and other nuclear antibodies), mixed connective tissue disease (often characterized by antibodies that extract nuclear antigens (such as ribonucleoproteins)), polymyositis/epidermatomyositis (often characterized by nongroup ANA), Pernicious anemia (usually characterized by antiparietal cell, microsomal, and intrinsic factor antibodies) primary Addison's disease (usually characterized by humoral and cell-mediated adrenal toxicity), infertility (usually characterized by antisperm antibodies), renal Glomerulonephritis (usually characterized by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus (usually characterized by IgG and compensation in the basement membrane), sjogren's Syndrome (often characterized by multitissue antibodies and/or nongroup ANA (SS-B)), diabetes (often characterized by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including asthma or cystic Fibrotic adrenergic drug resistance) (often characterized by beta adrenergic receptor antibodies).

Additional autoimmune diseases that can be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, chronic active hepatitis (usually characterized by smooth muscle antibodies), primary biliary cirrhosis (usually characterized by mitochondrial antibodies) , other endocrine gland lesions (often characterized by specific tissue antibodies in some cases), vitiligo (often characterized by melanocytic antibodies), vascular lesions (often characterized by intravascular Ig and complement and/or low serum complement), MI Sequelae (characterized by myocardial antibodies), postcardiac syndrome (usually characterized by myocardial antibodies), urticaria (usually characterized by IgG and IgM antibodies to IgE), atopic dermatitis (usually characterized by IgG and IgM antibodies to IgE) , asthma (often characterized by IgG and IgM antibodies to IgE), inflammatory myopathy and many other inflammatory, granulamatous, degenerative and other atrophic diseases.

In a preferred embodiment, the aforementioned autoimmune diseases and dysfunctional and/or pathological states are treated, prevented and/or diagnosed with anti-Neutrokine-α antibody and/or anti-Neutrokine-αSV antibody.

In a specific preferred embodiment, rheumatoid arthritis is treated, prevented and/or diagnosed with anti-Neutrokine-α antibodies and/or anti-Neutrokine-αSV antibodies and/or other antagonists of the invention.

In a specific preferred embodiment, lupus is treated, prevented and/or diagnosed with the anti-Neutrokine-α antibodies and/or Neutrokine-αSV antibodies and/or other antagonists of the invention.

In a specific preferred embodiment, lupus-associated nephritis is treated, prevented and/or diagnosed with the anti-Neutrokine-α antibody and/or Neutrokine-αSV antibody and/or other antagonists of the present invention.

In a specific embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or antagonists thereof (such as anti-Neutrokine-α antibodies and/or Neutrokine-αSV antibodies), are used to treat or prevent systemic Lupus erythematosus and/or diseases related thereto. Lupus-related diseases that can be treated with Neutrokine-α antibodies and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, or antagonists thereof, include but are not limited to blood diseases (such as hemolytic anemia, leukemia, lymphocytic anemia , and platelet anemia), immune disorders (eg, anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity, mouth sores, arthritis, fever, fatigue, weight loss, serous disorders (eg, pleurisy), renal disease (eg, nephritis), neuropathy (eg, episodic peripheral neuropathy, CNS-related disorders), gastrointestinal disorders, Raynaud's phenomenon, eg, pericarditis. In a preferred embodiment, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides, or antagonists thereof (such as anti-Neutrokine-α and/or Neutrokine-αSV antibodies), are used for treatment or prophylaxis with systemic Kidney disease associated with lupus erythematosus. In a more preferred embodiment, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or polypeptides, or antagonists thereof (such as anti-Neutrokine-alpha and/or Neutrokine-alphaSV antibodies), are used for treatment or prophylaxis with systemic Nephritis associated with lupus erythematosus.

Similarly, allergic diseases such as asthma (especially allergic asthma) or other respiratory diseases can also be treated with Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention . Additionally, these molecules can be used in the treatment, prevention and/or diagnosis of allergy, hypersensitivity, or blood group incompatibility to antigenic molecules.

Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, and/or agonists and/or antagonists thereof, can also be used for the treatment, prevention and/or diagnosis of organ rejection or transplant host rejection disease (GVHD) and/or diseases associated with it. Organ rejection occurs when the transplanted tissue destroys the host's immune cells through an immune response. Similarly, an immune response is also involved in GVHD, but in this case, exogenously transplanted immune cells destroy host tissues. Administration of Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists/antagonists thereof of the present invention that suppress immune responses, especially T cell proliferation, differentiation or chemotaxis, can effectively prevent organ rejection Or GVHD.

Similarly, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention may also be used to modulate inflammation. For example, Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention can inhibit the proliferation and differentiation of cells in response to inflammation. These molecules can be used in the treatment, prevention and/or diagnosis of acute and chronic inflammation, including chronic prostatitis, granulomatous prostatitis, and macera mata, inflammation associated with infection (such as septic shock, or systemic inflammatory response syndrome (SIRS) ), ischemia-reperfusion injury, endotoxin-lethal disease, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or Diseases due to overproduction of cytokines such as TNF or IL-1.

In a specific embodiment, the anti-Neutrokine-α and/or Neutrokine-αSV antibodies of the invention are used for the treatment, prevention, regulation, detection and/or diagnosis of inflammation.

In a specific embodiment, the anti-Neutrokine-α and/or Neutrokine-αSV antibodies of the invention are used for the treatment, prevention, regulation, detection and/or diagnosis of inflammatory diseases.

In another specific embodiment, the anti-Neutrokine-α and/or Neutrokine-αSV antibodies of the invention are used for the treatment, prevention, regulation, detection and/or diagnosis of allergic and/or hypersensitivity diseases.

Anti-Neutrokine-α and/or Neutrokine-αSV antibodies can be used to bind and inhibit Neutrokine-α and/or Neutrokine-αSV activity to treat, prevent and/or diagnose ARDS by preventing neutrophil infiltration of the lung after injury. The agonists and antagonists of the present invention may be used in combination with a pharmaceutically suitable carrier, as described hereinafter.

Neutrokine-α and/or Neutrokine-αSV and/or Neutrokine-α receptor polynucleotides or polypeptides of the present invention, and/or agonists and/or antagonists thereof, for the treatment, prevention and/or diagnosis of pulmonary diseases (such as bronchial diseases, such as sinopulmonary and bronchial infection, and related diseases and other respiratory diseases). In specific embodiments, such diseases include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (such as bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps, bronchiectasis ( eg, dry bronchiectasis, barrel bronchiectasis, and cystic bronchiectasis), bronchial adenocarcinoma, bronchial carcinoma, bronchiolitis (eg, exudative bronchiolitis, fibromatous bronchiolitis obliterans, and proliferative bronchiolitis) , broncho-alveolar carcinoma, bronchial asthma, bronchitis (eg, asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, crouping bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, occlusive bronchitis, plastic bronchitis, pseudomembranous bronchitis, septic bronchitis, and helminthic bronchitis), bronchocentral granulomatous disease, bronchial edema, bronchoesophageal disease, bronchogenic carcinoma, bronchogenic cyst , broncholithiasis, bronchomalacia, bronchomycoses (such as bronchopulmonary aspergillosis), bronchopulmonary spirochetes, hemorrhagic bronchitis, bronchial mucorrhea, bronchospasm, bronchial hemorrhage, bronchial stenosis, Biot's breath sounds, bronchial breath sounds, Kussmaul breath sounds, Kussmaul-kien breath sounds, respiratory acidosis, respiratory alkalosis, neonatal respiratory distress syndrome, respiratory insufficiency, respiratory sclerosis, respiratory syncytial virus, etc.

In a specific embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, and/or agonists and/or antagonists thereof, are used for the treatment, prevention, and/or diagnosis of chronic obstructive chronic pulmonary disease (COPD).

In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention are used for the treatment, prevention and/or diagnosis of fibrosis-related Lesions such as but not limited to cystic fibrosis (including pancreatic cystic fibrosis, Clarke-Hadfield syndrome, pancreatic fibrocyst, and mucinous lesions), myocardial fibrosis, primary retroperitoneal fibrosis, leptomyces Membranous fibrosis, mediastinal fibrosis, subepidermal nodular fibrosis, pericentral fibrosis, perimuscular fibrosis, vocal cord fibrosis, subadventitial fibrosis, and Symmer's clay fibrosis.

TNF family ligands are known to be the most potent cytokines, inducing a multitude of cellular responses, including cytotoxicity, antiviral activity, immunomodulatory activity, and transcriptional regulation of some genes (D.V. Goeddet et al., "Tumor necrosis factor: gene structure and biological Activity", Symp.Quant.Biol.51:597-609 (1986), Cold Spring Harbor; B.Beutler and A.Cerami, Biochemical Analytical Research 57:505-5181 (1988); L.J.Old.Sci.Am.258 : 59-75 (1988); W. Fiers, FEBs Communications 284: 199-224 (1991)). TNF family ligands, including Neutrokine-α and/or Neutrokine-αSV of the present invention, induce such various cellular responses by binding to TNF family receptors. Neutrokine-α and/or Neutrokine-αSV polypeptides are believed to elicit effective cellular responses, including cells, cell lines, tissues. Genotypic, phenotypic and/or morphological changes in tissue cultures or patients. As noted above, such cellular responses include not only normal physiological responses to TNF family ligands, but also diseases associated with increased or inhibited apoptosis. Apoptosis is a physiological mechanism involved in the loss of peripheral B and/or T lymphocytes of the immune system, and its dysfunction can lead to many different pathological changes (J.C.Ameisen, AIDS8:1197-1213, (1994); P.H. Krammer et al., General Opinion in Immunology 6:279-289 (1994)).

The Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention, and agonists and antagonists thereof can be used to diagnose, treat or prevent diseases related to the improvement of cell viability or the inhibition of apoptosis, including cancer (such as Cystic lymphoma, cancer caused by p53 mutation, and hormone-dependent tumors, including but not limited to colon cancer, cardiac tumors, pancreatic duct cancer, melanoma, retinal glioma, malignant glioma, lung cancer, intestinal Carcinoma, testicular cancer, bone cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastic C tumor, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (eg, systemic lupus erythematosus, and immune-related glomerulonephritis, rheumatoid arthritis); viral infections

(eg, herpesviruses, poxviruses, and adenoviruses); inflammation; graft-versus-host; acute and chronic graft rejection. Therefore, in a preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists or antagonists thereof of the present invention are used for the treatment, prevention and/or diagnosis of autoimmune diseases and And/or inhibit the growth, progression and/or metastasis of cancers, including but not limited to those cancers described herein, such as lymphocytic leukemia (including, for example, MLL, and chronic lymphocytic leukemia (CLL) and lymphoid follicle tumor. In another embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides of the present invention are used to activate cancer cells or tissues (such as B cell lineage-related cancers (such as CLL and MLL), lymphocytic leukemia , or lymphoma) to differentiate or proliferate, thereby making the cells more receptive to cancer treatments (such as chemotherapy or radiation).

In addition, in other embodiments, the Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention or its agonist or antagonist is used to inhibit the growth, progression and/or metastasis of malignant tumors and related diseases, Such diseases as leukemia (including acute leukemia (such as acute lymphoblastic leukemia), acute myelocytic leukemia (including myeloid C leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and Erythroleukemia)) and chronic leukemia (such as chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (Hodgkin's disease and non-Hdgkin's disease), multiple myeloma, Waldenstrom's macrocytosis Proteinemia, heavy chain disease, and solid tumors including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, myeloma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphovascular Endothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, Sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervix Carcinoma, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytic carcinoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, angioblastoma Cytoma, acoustic neuroma, oligodendroglioma, melanoma, neuroblastoma, and retinoblastoma.

The Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide and its agonists and antagonists of the present invention can be used to diagnose, treat or prevent diseases with increased apoptosis, including AIDS; neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis, cerebellar degeneration); myelodysplastic syndromes (eg, aplastic anemia), ischemic injury (eg, myocardial infarction, stroke, and reperfusion injury), toxin-induced Liver disease (eg, due to alcohol), septic shock, cachexia, and anorexia. Therefore, in a preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotides or polypeptides and/or agonists or antagonists thereof of the present invention are used for the treatment, prevention and/or diagnosis of the aforementioned diseases.

In a preferred embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention and/or agonists or antagonists thereof (such as anti-Neutrokine-α antibodies) inhibit human histiocytic lymphoma U in a dose-dependent manner. Growth of -937 cells. In another preferred embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptide of the present invention and/or its agonist or antagonist (such as anti-Neutrokine-α antibody) inhibit PC-3 cells, HT-29 cells , Hela cells, MCF-7 cells and A293 cells growth. In another more preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide of the present invention and/or its agonist (such as anti-Neutrokine-α antibody) are used to inhibit prostate cancer, colon cancer , growth, progression and/or metastasis of cervical and breast cancer.

Therefore, in another preferred embodiment, the present invention relates to a method for increasing apoptosis induced by TNF family ligands, comprising administering an effective amount of Neutrokine-α to cells expressing Neutrokine-α and/or Neutrokine-αSV receptors. Alpha and/or Neutrokine-αSV, or an agonist or antagonist thereof, increases or decreases Neutrokine-α and/or Neutrokine-αSV-mediated signaling. Preferably, the increased or decreased Neutrokine-α and/or Neutrokine-αSV mediated signaling can treat, prevent and/or diagnose diseases in which apoptosis is decreased or expression of cytokines and adsorbed molecules is decreased. Agonists or antagonists may include soluble forms of Neutrokine-α and/or Neutrokine-αSV and monoclonal antibodies directed against Neutrokine-α and/or Neutrokine-αSV polypeptides.

In another aspect, the present invention relates to a method for inhibiting apoptosis induced by TNF family ligands, which comprises administering an effective amount of a drug that can increase or decrease Neutrokine-α to cells expressing Neutrokine-α and/or Neutrokine-αSV receptors. Agonists or antagonists of alpha and/or Neutrokine-alpha SV-mediated signaling. Preferably, the increase or decrease of Neutrokine-α and/or Neutrokine-αSV mediated signaling can treat, prevent and/or diagnose diseases in which apoptosis or NF-KB expression is increased. Agonists or antagonists may include soluble forms of Neutrokine-α and/or Neutrokine-αSV and monoclonal antibodies directed against Neutrokine-α and/or Neutrokine-αSV polypeptides.

Since Neutrokine-α and/or Neutrokine-αSV belong to the TNF superfamily, this polypeptide should also regulate angiogenesis. In addition, since Neutrokine-α and/or Neutrokine-αSV inhibit immune cell function, this polypeptide has broad anti-inflammatory activity. Neutrokine-α and/or Neutrokine-αSV can be used as an anti-neoangiogenic agent for the treatment, prevention and/or diagnosis of solid tumors, by stimulating the invasion and activation of host defense cells such as cytotoxic T cells and macrophages, and by Inhibits tumor angiogenesis. Those skilled in the art can recognize other non-cancerous indications where vascular proliferation is undesirable. They can also be used to enhance host resistance against chronic and acute infections, eg against myobacterial infections by attracting and activating microbicidal leukocytes. Neutrokine-α and/or Neutrokine-αSV can also inhibit T cell proliferation by inhibiting IL-2 biosynthesis for the treatment of T cell-mediated autoimmune diseases and lymphocytic leukemia (including, for example, chronic lymphocytic leukemia (CLL) ). Neutrokine-α and/or Neutrokine-αSV may also be used to stimulate wound healing by recruiting connective tissue for debris removal and initiation of inflammatory cells. In the same way, Neutrokine-α and/or Neutrokine-αSV can also be used in the treatment, prevention and/or diagnosis of other fibrotic diseases, including liver cirrhosis, osteoarthritis and pulmonary fibrosis. Neutrokine-α and/or Neutrokine-αSV also increase the content of eosinophils, which have the unique ability to kill a large number of parasites that infect tissues, such as schistosomes, trichinella and roundworms. It can also be used to regulate hematopoiesis by modulating the activation and differentiation of various hematopoietic progenitors, for example the release of mature leukocytes from the bone marrow following chemotherapy, ie in stem cell transfer. Neutrokine-α and/or Neutrokine-αSV can also be used in the treatment, prevention and/or diagnosis of sepsis.

The polynucleotides or/or polypeptides and/or agonists and/or antagonists thereof of the present invention can be used for diagnosis and treatment or prevention of many diseases and/or pathological changes. Such diseases and pathological changes include but are not limited to cancers (such as immune cell-related cancers, breast cancer, prostate cancer, ovarian cancer, lymphoid follicular tumors, cancers associated with p53 mutations or alterations, brain tumors, bladder cancer, cervical cancer , colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, etc.), lymphoproliferative diseases (such as lymph gland lesions), microbial (such as viruses, bacteria, etc.) infections, (such as HIV-1 infection, HIV -2 infection, herpes virus infection (including but not limited to HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis Viral infection (such as HAV, HBV, HCV, etc.), Helicobacter pylori infection, Staphylococcus, etc.), parasitic infection, nephritis, bone disease (such as osteoporosis), atherosclerosis, pain, cardiovascular disease (such as Neovascularization, decreased angiogenesis, or decreased circulating blood volume (such as ischemic diseases such as myocardial infarction, stroke, etc.)), AIDS, allergies, inflammation, neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, muscular atrophy Lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, etc.), transplant rejection (acute and chronic), graft-versus-host disease, diseases caused by bone marrow dysplasia (such as aplastic anemia, etc.), joint tissue destruction in rheumatic diseases , liver diseases (such as acute and chronic hepatitis, liver injury and cirrhosis), autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, Autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy (eg, dilated cardiomyopathy), diabetes, diabetic complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza , asthma, psoriasis, glomerulonephritis, septic shock and ulcerative colitis.

The polynucleotides and/or polypeptides of the present invention and/or their agonists and/or antagonists are used to promote angiogenesis and wound healing (such as wounds, burns and fractures). The polynucleotides and/or polypeptides of the present invention and/or their agonists and/or antagonists can also be used as adjuvants to enhance immune responses to specific antigens and antiviral immune responses.

Typically, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are used to modulate (ie stimulate or decrease) an immune response. For example, polynucleotides and/or polypeptides of the invention are useful in preparing for or recovering from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or in aiding immune response and/or recovery in the elderly and immunocompromised. Alternatively, the polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are used as immunosuppressants, for example in the treatment or prevention of autoimmune diseases. In specific embodiments, the polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune diseases, such as those described herein and others known in the art.

Preferably, with Neutrokine-α and/or Neutrokine-αSV polynucleotide or polypeptide, and/or its agonist or antagonist (such as anti-Neutrokine-α antibody), an effective amount of Neutrokine-α of the present invention and/or Either Neutrokine-αSV polypeptide, or an agonist or antagonist thereof, is administered to the patient; or cells are removed from the patient, the cells are fed with Neutrokine-α and/or Neutrokine-αSV polynucleotide, and the engineered cells are then readministered In patients [from the body treatment]. Additionally, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides may be used as adjuvants in vaccines as further described herein to generate an immune response against an infectious disease.

Formulation and Application Method

Considering the clinical conditions of each patient (especially only the side effects of using Neutrokine-α and/or Neutrokine-αSV polypeptide), the delivery location of Neutrokine-α and/or polypeptide composition, the method of administration, the procedure of administration, and other known factors, for For good medical use, Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptide compositions (preferably containing polypeptides that are soluble forms of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV ectodomain) are formulated, and dosed. An "effective amount" of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide is thus determined by this condition.

Generally, the total pharmaceutically effective amount per dose of Neutrokine-α and/or Neutrokine-αSV polypeptide administered parenterally ranges from approximately 1 μg/kg day to 10 mg/kg body weight/day, although therapeutic decisions will be made as described above . Preferably the dosage is at least 0.01 mg/kg body weight/day, more preferably between 0.01-1 mg/kg body weight/day.

In another embodiment, the dose of Neutrokine-α and/or Neutrokine-αSV polypeptide of the present invention administered to humans is between 0.0001-0.045 mg/kg body weight/day, preferably between 0.0045-0.045 mg/kg body weight/day between, more preferably 45 μg/kg body weight/day; the dose administered to mice is about 3 mg/kg body weight/day.

If applied continuously, Neutrokine-α and/or Neutrokine-αSV polypeptides are administered at doses of about 1 μg/kg body weight/hour to 50 μg/kg body weight/hour, injected 1-4 times a day or continuously subcutaneously infused, eg, with a micropump. Intravenous fluids may also be used.

The treatment time is determined according to the effect.

In a specific embodiment, the total pharmaceutically effective amount of each dose of Neutrokine-α and/or Neutrokine-αSV polypeptide for parenteral administration is in the range of about 0.1 μg/kg body weight/day to 45 μg/g body weight/day, although as stated above to make a therapeutic decision. Preferably, the dose is at least 0.1 μg/kg body weight/day, more preferably the dose administered to humans is between 0.01-50 μg/kg body weight/day. Neutrokine-α and/or Neutrokine-αSV can be infused continuously, multiple injections per day (such as three or more times a day, or twice a day, once a day, or intermittent injections (such as twice a day, once a day, every other day) Once, twice a week, once a week, once every two weeks, once a month, once a month, once a month). If applied continuously, the dose of Neutrokine-α and/or Neutrokine-αSV polypeptide is about 0.001-10 μg/kg body weight /hour to about 50 μg/kg body weight/hour, 1-4 injections per day or continuous subcutaneous infusion, eg using a micropump.

Effective doses of compositions of the invention administered can be determined by methods known in the art, noting parameters such as biological half-life, biological potency, and toxicity. Such assays are well known to those skilled in the art.

The biological exposure of the body to Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides during treatment also plays an important role in the determination and/or pharmaceutically effective amount. Dosage changes, such as repeated administration of relatively low doses of Neutrokine-α and/or Neutrokine-αSV polypeptides for relatively long periods of time, and repeated administration of relatively high doses of Neutrokine-α and/or Neutrokine-αSV polypeptides for relatively short periods of time, have Different therapeutic and/or pharmacological effects. See, eg, the assay for serum immunoglobulin levels shown in Example 6.

Using the equivalent surface area dose conversion factors provided by Freireich, E.J. et al. (Cancer Chemotherapy Reports 50 (4): 219-44 (1966)), those skilled in the art can routinely use Neutrokine-α and/or Neutrokine-αSV in Data from a given test are converted to an accurate estimate of the pharmaceutically effective amount of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide administered per dose in another test line. By administering the experimental data obtained from Neutrokine-α in mice (see for example Example 6), the conversion factor provided by Freireich et al. can be used to accurately assess the drug effectiveness of Neutrokine-α in rats, monkeys, dogs and humans. quantity. The following conversion table (Table 3) summarizes the data provided by Freireich et al. Table 3 provides approximate factors for converting doses expressed in mg/kg body weight in one species to equivalent surface area doses expressed in mg/kg body weight in another species.

Table 3 Equivalent surface area dose conversion factors

Thus, for example, using the conversion factors provided in Table 3, a dose of 50 mg/kg in mice converts to an appropriate dose of 12.5 mg/kg in monkeys because (50 mg/kg) x 1/4 = 12.5 mg/kg kg. Additionally, doses of 0.02, 0.08, 0.8, 2 and 8 mg/kg in mice were equivalent to 1.667 μg/kg, 6.67 μg/kg, 66.7 μg/kg, 166.7 μg/kg and 0.667 mg/kg in humans The effect of dosage.

Pharmaceutical compositions containing Neutrokine-α and/or Neutrokine-αSV polypeptides of the present invention can be administered orally, rectally, parenterally, subcutaneously, intracistemally, intravaginally, intraperitoneally, topically (by powder, ointment, drops or patch form) ), buccal, or via oral or nasal spray (such as steam or powder inhalation) and the like. In one embodiment, "pharmaceutically suitable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating substance or formulation auxiliary of any type. In a specific embodiment, "pharmaceutically suitable" refers to an article that is approved for use in animals, especially humans, by the US FDA, the regulatory agency. Suitable pharmaceutical carriers according to this embodiment are provided, for example without limitation, by E.W. Martin in "Remington's Pharmaceutical Sciences" and include sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as Peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is injected intravenously. Saline solutions and dextrose and glycerol solutions can be employed as liquid carriers, especially for injectable solutions, the compositions, if desired, also containing traces of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

The term "parenteral" refers to modes of administration including intravenous, intraperitoneal, subcutaneous and intraarterial and gluteal injection and infusion.

In a preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV compositions (including polypeptides, polynucleotides and antibodies, agonists and/or antagonists thereof) of the present invention are administered subcutaneously.

In another preferred embodiment, the Neutrokine-α and/or Neutrokine-αSV compositions (including polypeptides, polynucleotides and antibodies, agonists and/or antagonists thereof) of the present invention are administered intravenously.

The Neutrokine-α and/or Neutrokine-αSV compositions of the present invention may also be suitably administered by a sustained release system. Sustained release compositions include, for example, suitable polymers (such as semipermeable polymeric matrices, such as films or microcapsules), suitable hydrophobes (such as suitable oil emulsions), or ion exchange resins, and small amounts of soluble derivatives (such as small amounts of soluble salts).

Sustained-release matrices include polylactic acid (US Patent No.3773919, EP58481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman, V. et al., Biopolymers 22:547- 556 (1983)), poly(ethyl-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981)), and R. Langer. Chem. Tech. 12 : 98-105 (1982), ethyl vinyl acetate (R. Langer et al., Id.) or poly-D-3-hydroxybutyric acid (EP 133988).

Sustained release compositions also include compositions of the invention entrapped in liposomes (see Langer, Science 249:1527-1533 (1990)); Treat et al., Liposomes in Infectious Disease and Cancer Therapy, Lopez-Berestein and Fidler (editor), Liss, New York, pp. 317-327 and 353-365 (1989). Liposomes containing Neutrokine-α and/or Neutrokine-αSV polypeptides can be prepared by known methods, see DE 3218121; Epstein et al., PNAS 82:3688-3692 (1985); Hwang et al., PNAS 77 EP 52322; EP36676; EP88046; EP143949; EP142641; Japanese Patent Application 83-118008; Typically, liposomes are small monoliths (approximately 200-800 Angstroms) with a lipid content greater than 30 mol cholesterol, and the selection ratio is adjusted to optimize Neutrokine-α and/or Neutrokine-αSV polypeptide therapy.

In another embodiment, the sustained release compositions of the present invention include crystalline formulations known in the art.

In another embodiment, the composition of the present invention is delivered by a pump (see Langer, supra; Sefton, CRC Crit Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980) ; Saudek et al., N. Engl. J. Med 321:574 (1989)).

Other controlled release systems are described by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, Neutrokine-α and/or Neutrokine-αSV polypeptides are generally in the desired purity, in a unit dosage injectable form (solution, suspension or emulsion), together with a pharmaceutically suitable carrier , that is, a carrier that is non-toxic to the receptor and compatible with other ingredients in the formulation at the applied dose and concentration. For example, the formulation preferably does not include oxidizing agents and other compounds known to be detrimental to polypeptides.

Generally, the formulations are prepared by uniformly and intimately contacting Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide with liquid carriers or well-separated solid carriers or both. Then, if necessary, the product is formed into the desired shape. Preferably, the vehicle is a parenteral vehicle, more preferably a solution isotonic with the blood of the recipient. Such vehicles include, for example, water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed lipids and ethyl oleic acid and liposomes are also useful herein.

The carrier suitably contains minor amounts of additives such as to enhance isotonicity and chemical stability. Such substances are nontoxic to receptors at applied doses and concentrations, including buffers such as phosphoric acid, citric acid, succinic acid, acetic acid and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) ) polypeptides such as polyarginine or tripeptide; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; Monosaccharides, disaccharides or other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium ions; Preservatives such as cresol, phenol, chlorobutanol, benzyl alcohol, and parabens and/or nonionic surfactants such as polysorbates, poloxamers or PEG.

The concentration of Neutrokine-α and/or Neutrokine-αSV polypeptide formulated in this carrier is about 0.001mg/ml～100mg/ml, or 0.1mg/ml～100mg/ml, preferably 1-10mg/ml, or 1- 10mg/ml, the pH is about 3-10, or 2-8, preferably 5-8, more preferably 6-7. It will be appreciated that use of some of the foregoing receptors, carriers or stabilizers will result in the formation of salts of Neutrokine-alpha and/or Neutrokine-alphaSV.

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides for therapeutic administration must be sterile. Sterility can be achieved by filtration through a sterile filter membrane (eg, a 0.2 micron membrane). Therapeutic Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptide compositions are generally placed in containers with sterile access, such as intravenous solution packs or vials with stoppers that can be penetrated by hypodermic needles.

Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, in aqueous solution or in reconstitutable lyophilized form. The lyophilized formulation is, for example, a 10 ml vial filled with 5 ml sterile filtered 1% (w/v) Neutrokine-α and/or Neutrokine-αSV polypeptide aqueous solution, and the resulting mixture is lyophilized. Perfusate is prepared by reconstituting lyophilized Neutrokine-α and/or Neutrokine-αSV polypeptide with sterile water for injection.

Alternatively, Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides are stored in lyophilized form in single-dose containers. This perfusate is reconstituted with a sterile injectable vehicle.

The invention also provides a pharmaceutical package or kit. Comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. This container is also accompanied by a notice of approval for use in humans by the government agency responsible for the approval of the manufacture, use or sale of pharmaceutical or biological products. In addition, the polypeptides of the present invention may be used in combination with other therapeutic compounds.

The compositions of the invention may be administered alone or in combination with other adjuvants. Adjuvants that may be used in combination with the compositions of the present invention include, but are not limited to, alum, alum plus deoxycholate (Immuno Ag), MTP-PE (Biocine Corp.) QS21 (Genentech, Inc.), BCG and MPI . In a specific embodiment, the composition of the invention is administered in combination with QS-21. Other adjuvants that may be used in combination with the compositions of the present invention include, but are not limited to, monophosphate lipid immunomodulators, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts, MF-59, and viral adjuvants. Vaccines that can be used with the composition of the present invention include, but are not limited to, MMR (measles, mumps, rubella) vaccine, polio vaccine, varicella vaccine, tetanus vaccine, diphtheria vaccine, hepatitis A vaccine, hepatitis B vaccine, Haemophilus Influenza B vaccine, pertussis vaccine, pneumonia vaccine, influenza vaccine, Lyme's disease vaccine, cholera vaccine, yellow fever vaccine, meningitis vaccine, rabies vaccine, typhoid fever vaccine, whooping cough vaccine and/or PNEUMOVAX-23 ^TM . Can be administered in concomitant combination, such as as an admixture, separately but simultaneously; or can be administered in sequential combination, which includes presentation where the combination agents are administered together as a therapeutic mixture, also includes the combination agents separately but simultaneously, such as via separate intravenous access into the same body. "Combined" administration also includes the separate administration of one compound followed by the other.

In another specific embodiment, the composition of the present invention is used in combination with PNEVMOVAX-23TM for the treatment, prevention and/or diagnosis of infectious diseases and/or any diseases associated therewith. In one embodiment, the composition of the present invention is administered in combination with PNEVMOVAX-23 ^™ for the treatment, prevention and/or diagnosis of any Gram-positive bacterial infection and/or any disease associated therewith. In another embodiment, a composition of the present invention is administered in combination with PNEVMOVAX-23 ^(TM) for the prophylaxis and/or diagnosis of infections associated with members of Enterobacter and/or Streptomyces. In another embodiment, the composition of the present invention is used in combination with PNEOMOVAX-23 ^(TM) to treat, prevent and/or diagnose diseases associated with one or more members of the B Streptococcus genus. In another embodiment, the composition of the present invention is used in combination with PNEOMOVAX-23 ^™ to treat, prevent and/or diagnose diseases associated with Streptococcus pneumoniae.

The compositions of the present invention may be administered alone, or in combination with other therapeutic agents, including but not limited to chemotherapeutics, antibiotics, antivirals, steroidal and nonsteroidal anti-inflammatory agents, conventional immunotherapeutics and cytokines. Combination administration may be concomitant administration eg as a mixture, separate but simultaneous administration; or sequential administration. It includes the administration of the combination agents together as a therapeutic mixture, and also includes the administration of the combination agents separately but simultaneously, such as simultaneous infusion into the same individual through separate intravenous accesses, and "combination" administration also includes the separate administration of one compound followed by the other. kind.

In one embodiment, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered in combination with the compositions of the present invention include, but are not limited to, soluble forms of TNF-α and/or α-lymphotoxin (α-LT, also known as TNF-β), LT -β (found in the heterotrimeric LT-α2-β complex), OPGL, FasL, CD27L, CD30L, CD40L, 40-1BBL, DcR3, OX40L, γ-TNF (International Publication WO 96/14328) , AIM-I (International Publication WO 97/33899), AIM-II (International Publication WO 97/34911), APRIL (J.Exp.Med.188 (6): 1185-1190), α-intrinsic factor (International Publication WO98/07880), TR6 (International Publication WO 98/30694), OPG and Neutrokine-α (International Publication WO 98/18921), OX40 and nerve growth factor (NGF) and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication WO98/34095), DR3 (International Publication WO97/33904), DR4 (International Publication WO98/32856), TR5 (International Publication WO98/30693), TR6 (International Publication WO98/30694) TR7 (International Publication WO 98/41629), TRANK, TR9 (International Publication WO 98/56892), TR10 (International Publication WO 98/54202), 312C2 (International Publication WO 98/06842) and TR12.

In a preferred embodiment, the compositions of the present invention are combined with CD40 ligand (CD40L), soluble forms of CD40L (such as AVREND ^™ ), biologically active fragments, variants or derivatives of CD40L, anti-CD40L antibodies (such as agonist Antibodies or antagonistic antibodies), and/or anti-CD40 antibodies (such as agonistic or antagonistic antibodies).

In some embodiments, compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptases that may be administered in combination with the compositions of the present invention include, but are not limited to, RETROVIR ^™ (zidovudine/AZT), VIDEX ^™ (didanosine/ddI), HIVID ^™ (zalcitabine/ddC), ZERIT ^™ (stavudine/d4T), EPIVIR ^™ (lamivudine/3TC), and COMBIVIR ^™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptases that can be administered in combination with the compositions of the invention include, but are not limited to, VIRAMUNE ^™ (nevirapine), RESCRIPTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirenz). Protease inhibitors that can be administered in combination with the compositions of the present invention include, but are not limited to, CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir), INVIRASE ^™ (saquinavir), and VIRACEPT ^™ (nelfinavir). In a specific embodiment, antiviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be combined with the compositions of the present invention in any combination, therapeutically, prophylactically and/or diagnostically AIDS and/or treatment, prevention and/or diagnosis of HIV infection.

In other embodiments, the compositions of the present invention may be used in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the compositions of the present invention include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , ATOVAQUONE ^™ , ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ , RIFABUTIN ^{™ ,} CLARITHROMAZITH ROM, ^TM , ^{GANCICLOVIRTM} , ^FOSCARNETTM , ^CIDOFOVIRTM , ^{FLUCONAZOLETM} , ^{ITRACONAZOLETM} , KETOCONAZOLETM, ^ACYCLOVIRTM , ^{FAMCICOLVIRTM , PYRIMETHAMINETM} , ^{LEUCOVORINTM ,} NEVPOGENTM ⁽ filgrastim-img/LEGINE, CSF). In a specific embodiment, the composition of the present invention can be freely combined with TRIMETHOPRIM-SULFAMETHOXAZOLE ^TM , DAPSONE ^TM , PENTAMIDINE ^TM and/or ATOVAQUONE ^TM for prophylactic treatment, prevention and/or diagnosis of opportunistic Pneumocystis carinii infection. In another specific embodiment, the composition of the present invention is optionally combined with ISONIAZID ^TM , RIFAMPIN ^TM , PYRAZINAMIDE ^TM and/or ETHAMBUTOL ^TM for prophylactic treatment, prevention and/or diagnosis of opportunistic Mycobacterium avium complex infection. In another specific embodiment, the composition of the present invention is optionally combined with RIFABUTIN ^TM , CLARITHROMYCIN ^TM and AZITHROMYCIN ^TM for prophylactic treatment, prevention and/or diagnosis of opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, the composition of the present invention is optionally combined with GANCICLOVIR ^™ , FOSCARNET ^™ and/or CIDOFOVIR ^™ for prophylactic treatment, prevention and/or diagnosis of opportunistic cytomegalovirus infection. In another specific embodiment, the composition of the present invention is optionally combined with FLUCONAZOLE ^™ , ITRACONAZOLE ^™ , and/or KETOCONAZOLE ^™ for the prophylactic treatment, prevention and/or diagnosis of opportunistic fungal infections. In another specific embodiment, the composition of the present invention is optionally combined with ACYCOLOVIR ^™ and/or FAMCICOLVIR ^™ for prophylactic treatment, prophylaxis and/or diagnosis of opportunistic Type I or Type II herpes simplex virus infection. In another specific embodiment, the composition of the present invention is optionally combined with PYRIMETHAMINE ^™ and/or LEUCOVORIN ^™ for prophylactic treatment, prevention and/or diagnosis of opportunistic Toxoplasma gondii infection. In another specific embodiment, the composition of the present invention is optionally combined with LEUCOVORIN ^™ and/or NEUPOGEN ^™ for the prophylactic treatment, prevention and/or diagnosis of opportunistic bacterial infections.

In another embodiment, the composition of the invention is administered in combination with an antiviral agent. Antiviral agents that may be administered in combination with the compositions of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In another embodiment, the compositions of the invention are administered in combination with antibiotics. Antibiotics that may be administered in combination with the compositions of the present invention include, but are not limited to, amoxicillin, aminoglycoside antibiotics, beta-lactams (glycopeptides), beta-lactamase, clindamycin, chloramphenicol, cephalosporins , ciprofloxacin, erythromycin, fluoroquinomycin, macrolide, 2-methyl-5-nitro-1-imidazolyl ethanol, penicillin, quinomycin, rifampicin, streptomycin, ammonia Sulfonyl, tetracycline, trimethoprim, trimethoprim-thiamine, and vancomycin.

Conventional non-specific immunosuppressants that may be administered in combination with the compositions of this invention include, but are not limited to, steroids, cyclosporine, the cyclosporine analog cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, The pine dragon, azathioprine, FK-506, 15-deoxy spergualin and other immunosuppressants that suppress the function of responding T cells.

In a specific embodiment, the compositions of the invention are administered in combination with immunosuppressants. Immunosuppressant preparations that can be administered in combination with the compositions of the present invention include, but are not limited to, ORTHOCLONE ^™ (OKT3), SANDIMMUNE ^™ /NEORAL ^™ /SANGDYA ^™ (cyclosporin), PROGRAF ^™ (tacrolimus), CELLCEPT ^™ (mycophenolate Azathioprine), azathioprine, glucorticosteroids, and RAPAMUNE ^TM (sirolimus). In a specific embodiment, immunosuppressants are used to prevent rejection of organ or bone marrow transplants.

In a preferred embodiment, the compositions of the invention are administered in combination with steroid therapy. Steroids that may be used in combination with the present invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisone (eg, IV methylprednisone). In a specific embodiment, the composition of the invention is administered in combination with prednisone. In another specific embodiment, the composition of the invention is administered in combination with prednisone and an immunosuppressant. Immunosuppressants that can be administered in combination with the compositions of the invention and prednisone are those described herein, including but not limited to azathioprine, cyclophosphamide and cyclophosphamide IV. In another specific embodiment, the composition according to the invention is administered in combination with methylprednisone. In another specific embodiment, the composition of the invention is used in combination with methylprednisone and an immunosuppressant. Immunosuppressants that can be administered in combination with the compositions of the invention and methylprednisone are those described herein, including but not limited to azathioprine, cyclophosphamide and cyclophosphamide IV.

In a preferred embodiment, the composition of the invention is administered in combination with an antimalarial agent. Antimalarial agents that may be administered in combination with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the invention are administered in combination with NSAIDs.

In one embodiment, the composition of the present invention is administered in combination with one, two, three, four, five, ten or more of the following drugs: NRD-101 (Hoechst Marion Roussel), Cyclofluoperazine (Dimethaid), Oxapro Potassium azine (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco ), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelonemesylate (Warner-Lambert), soluble TNF receptor 1 (Synergen; Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), Leuko Vax (Inflammatics), MK-663 (Merch), ST-1482 (Sigma-Tau), and butixocort propionate (Warner Lamb bent).

In a preferred embodiment, the composition of the present invention is administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, azosulfapyridine salicylate, sodium aurothiomalate, thioglucose GOLD, cyclosporine, penicillamine, azathioprine, antimalarials (as described herein), cyclophosphamide, cyclosporine, gold, ENBREL ^TM (Etanercept), anti-TNF antibodies, and prednisolone .

In a more preferred embodiment, the composition of the present invention is administered in combination with antimalarial drugs, methotrexate, anti-TNF antibodies, ENBREL ^™ and/or azosulfapyridine salicylate. In one embodiment, the composition of the invention is administered in combination with methotrexate. In another embodiment, a composition of the invention is administered in combination with an anti-TNF antibody. In another embodiment, a composition of the invention is administered in combination with methotrexate and an anti-TNF antibody. In another embodiment, the composition of the invention is administered in combination with azosulfapyridine salicylate. In another specific embodiment, the compositions of the invention are administered in combination with ENBREL ^™ . In another embodiment, a composition of the invention is administered in combination with ENBREL ^™ , methotrexate, and azosulfapyridine salicylate. In other embodiments, one or more antimalarial drugs are administered in combination with one of the combinations described above. In a specific embodiment, a composition of the invention is administered in combination with an antimalarial drug (such as hydroxychloroquine), azosulfapyridine salicylate, an anti-TNF antibody, and methotrexate.

In another embodiment, the compositions of the invention are administered alone or in combination with one or more intravenously injected immunoglobulins. Intravenous immunoglobulins that may be administered in combination with the compositions of the present invention include, but are not limited to, GAMMAR ^™ , IVEEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S/D ^™ , and GAMIMUNE ^™ . In a specific embodiment, the compositions of the invention are administered in combination with intravenous immunoglobulin in transplantation therapy (eg, bone marrow transplantation).

CD40 ligand (CD40L), a soluble form of CD40L (such as AUREND ^™ ), biologically active fragments, variants or derivatives of CD40L, anti-CD40L antibodies (such as agonistic or antagonistic antibodies), and/or anti-CD40 Antibodies (such as agonistic or antagonistic antibodies).

In another embodiment, the composition of the invention is administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered in combination with the compositions of the present invention include, but are not limited to, glucocorticoids and non-steroidal anti-inflammatory agents, aminoaryl carboxylic acid derivatives, aryl acetic acid derivatives, aryl butyric acid derivatives, aryl Butyric acid derivatives, aryl carboxylic acid derivatives, aryl propionic acid derivatives, pyrazole, pyrazoline, salicylic acid derivatives, carbamoylthiazine, e-acetylaminocaproic acid, S-adenosylmethazine thionine, 3-amino-4-hydroxybutyric acid, amixetrine, phenylindole, 6-benzyladenine, bucolon, bisphenidamine, bibenconazole, emorfazone, guaiacol, naptine Metone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxine, proquazone, proxazole, and tenidapa.

In another embodiment, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered in combination with the compositions of the present invention include, but are not limited to, antibiotic derivatives (such as doxorubicin, bleomycin, daunorubicin, and daunorubicin); antiestrogens (such as tamoxifen); Antimetabolites (eg, fluorouracil, 5-FU, methotrexate, 5-fluorouracil deoxynucleoside, alpha-interferon 2b, glutamic acid, mithramycin, mercaptopurine, and b-thioguanine); cytotoxic agents ( Such as carmustine, BCNU, 1-cyclohexylnitrosourea, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine phosphate, diethylestradiol, estradiol, progesterone acetate, methyltestosterone, diethylstilbestrol diphosphate, trimethoxyphene vinyl chloride, and testolactone); nitrogen mustard derivatives (such as mephalen, chorambucil, mechlorethamine (nitrogen mustard) and triamidophos); steroids and combinations (such as bethamethasone sodium phosphate); and other substances (such as dicarbazine, dicarbazine, paraginase, mitotane, vincristine sulfate, vinblastine sulfate, and epipodophyllotoxin glucopyranoside).

In a specific embodiment, the composition of the invention is administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or with any combination of CHOP components. In another embodiment, the composition of the invention is administered in combination with Rituximab. In another embodiment, the composition of the invention is administered in combination with Rituximab and CHOP, or any combination of Rituxamab and CHOP components.

In another embodiment, the compositions of the invention are administered in combination with cytokines. Cytokines that may be administered in combination with the compositions of the present invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN- α, IFN-β, IFN-γ, TNF-α, TNF-β. In another embodiment, the compositions of the invention may be administered in combination with any interleukin, including but not limited to IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL -6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 , IL-19, IL-20, IL-21 and I1-22. In a preferred embodiment, the composition of the invention is administered in combination with IL4 and IL10. The inventors observed that both IL4 and IL10 enhance Neutrokine-α-mediated B cell proliferation.

In another embodiment, the compositions of the invention are administered in combination with chemokines. In another embodiment, a composition of the invention is administered in combination with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein 4. In a preferred embodiment, the composition of the invention is administered in combination with the chemokine beta-8.

In another embodiment, the compositions of the invention are administered in combination with an IL-4 antagonist. IL-4 antagonists that can be administered in combination with the compositions of the present invention include, but are not limited to, soluble IL-4 receptor polypeptides, soluble IL-4 receptor polypeptides in multimeric form; bind IL-4 receptor but do not transduce Anti-IL-4 receptor antibodies that induce biological signals elicited by IL-4; anti-IL-4 antibodies that block IL-4 binding to one or more IL4 receptors, and bind IL4 receptors but do not transduce IL4 IL4 muteins stimulate biological signals. Preferably, the antibodies used according to this method are monoclonal antibodies (including antibody fragments, as described herein).

In another embodiment, the compositions of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered in combination with the compositions of the present invention include, but are not limited to, LEUKINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FILGRASTIM ^™ ).

In another embodiment, the composition of the invention is administered in combination with fibroblast growth factors. Fibroblast growth factors that may be administered in combination with the compositions of the present invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In addition, the compositions of the present invention may be administered alone or in combination with other therapeutic methods, including but not limited to radiation therapy. Such combination therapy may be administered sequentially and/or concomitantly.

Agonists and Antagonists - Analysis and Molecules

The present invention also provides a method for screening compounds to identify enhancing or blocking effects of Neutrokine-α and/or Neutrokine-αSV polypeptides on cells, such as binding molecules such as receptor molecules to Neutrokine-α and/or Neutrokine-αSV interacting compounds. An agonist is a compound that enhances the natural biological function of Neutrokine-α and/or Neutrokine-αSV, or functions in a manner similar to Neutrokine-α and/or Neutrokine-αSV, while an antagonist is a compound that reduces or eliminates this function.

In another embodiment, the present invention provides a method of specifically binding to a receptor protein or other ligand binding protein of Neutrokine-α and/or Neutrokine-αSV polypeptide. For example, cellular compartments, such as cell membranes or preparations thereof, can be prepared from cells expressing molecules that bind Neutrokine-α and/or Neutrokine-αSV. This preparation is incubated with labeled Neutrokine-α and/or Neutrokine-αSV, complexes of Neutrokine-α and/or Neutrokine-αSV or other binding proteins bound to the receptor are isolated and characterized according to routine methods known in the art . Alternatively, Neutrokine-α and/or Neutrokine-αSV can be combined with a solid support, so that the binding molecules lysed from cells can be combined with a chromatography column, and then eluted and characterized according to conventional methods.

In the agonist or antagonist assays of the present invention, cellular compartments, such as cell membranes or preparations thereof, may be prepared from cells expressing molecules that bind Neutrokine-α and/or Neutrokine-αSV, such as through Neutrokine-α and/or or Neutrokine-αSV modulated signaling or regulatory pathway molecules. This preparation is incubated with labeled Neutrokine-[alpha] and/or Neutrokine-[alpha]SV in the presence or absence of a candidate molecule, which may be an agonist or antagonist of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV. The ability of the candidate molecule to bind to the binding molecule is reflected in a decreased binding of the labeled ligand. Unilaterally binding molecules, ie molecules that do not induce the effect of Neutrokine-α on the binding of Neutrokine-α and/or Neutrokine-αSV binding molecules, are good antagonists. Molecules that bind well and elicit actions similar or closely related to Neutrokine-α and/or Neutrokine-αSV are agonists.

Potential agonists and antagonists of Neutrokine-α and/or Neutrokine-αSV-like effects can be determined, for example, by determining the activity of the second messenger system after the candidate molecule interacts with cells or appropriate cell preparations, and interacts with Neutrokine-α and/or Neutrokine-αSV or a molecule that elicits the same effect as Neutrokine-α and/or Neutrokine-αSV is determined in comparison. Second messenger systems useful herein include, but are not limited to, AMP guanylate cyclase, ion channels, or phosphohydrolytic second messenger systems.

Another assay for Neutrokine-α and/or αSV antagonists is, for example, a competition assay, which is a combination of Neutrokine-α and/or Neutrokine-αSV and a potential antagonist, membrane-bound receptor molecule or Competitive inhibition assays were performed with recombinant Neutrokine-α and/or Neutrokine-αSV receptor molecules. Neutrokine-alpha and/or Neutrokine-alphaSV can be labeled eg by radioactivity so that the number of Neutrokine-alpha and/or Neutrokine-alphaSV molecules bound to the receptor molecule can be accurately determined to confirm the potency of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides (eg, IL-13) and antibodies that bind to the polypeptides of the invention, thereby inhibiting or extinguishing their activity. Potential antagonists can also be small organic molecules, peptides, polypeptides such as closely related proteins or antibodies that bind at the same site on a binding molecule such as a receptor molecule, without inducing Neutrokine-α and/or Neutrokine-αSV-induced activity, The action of Neutrokine-α and/or Neutrokine-αSV is thereby prevented by blocking the binding of Neutrokine-α and/or Neutrokine-αSV.

Other potential antagonists include antisense molecules. Antisense methods can be used to control gene expression by antisense DNA or RNA or by triple helix formation. Antisense methods are eg described by Okara, J. Neurochemical 56:560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression", CRC Press, Boca Raton, F1 (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991). This method is based on the binding of polynucleotides to complementary DNA or RNA. For example, the 5' coding portion of a polynucleotide encoding the ectodomain of a polypeptide of the present invention can be used to design antisense RNA oligonucleotides approximately 10-40 base pairs in length. Design a DNA oligonucleotide complementary to the region of the gene involved in the transcription, thereby preventing the transcription and production of Neutrokine-α and/or Neutrokine-αSV. This antisense RNA oligonucleotide hybridizes to mRNA in vivo and blocks translation of mRNA into Neutrokine-α and/or Neutrokine-αSV polypeptide. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of Neutrokine-α and/or Neutrokine-αSV.

In one embodiment, the Neutrokine-[alpha] and/or Neutrokine-[alpha]SV antisense nucleic acids of the invention are produced intracellularly by transcription from exogenous sequences. For example, a vector or a portion thereof that is transcribed to produce an antisense nucleic acid (RNA) of the invention. Such vectors contain sequences encoding Neutrokine-α and/or Neutrokine-αSV antisense nucleic acids. This vector can remain episomal or become a chromosomally integrated vector until it can be transcribed to produce the desired antisense DNA. Such vectors can be constructed by recombinant DNA methods standard in the art. Vectors can be plasmids, viruses, or other substances known in the art for replication and expression in vertebrate cells. Sequences encoding Neutrokine-α and/or Neutrokine-αSV, or fragments thereof, can be expressed in vertebrate, especially human cells, by any promoter known in the art. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980)), the herpes thymidine promoter (Wagner et al., PNAS 78:1441-1445 (1981), the regulatory sequence of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982) ),wait.

The antisense nucleic acid of the present invention comprises a sequence complementary to at least a portion of the RNA transcript of the Neutrokine-α and/or Neutrokine-αSV gene. However, absolute complementarity, while preferred, is not required. The sequence "complementary to at least a part of RNA" refers to a sequence that has good complementarity and can hybridize with RNA to form a stable duplex; in the case of double-stranded Neutrokine-α and/or Neutrokine-αSV antisense nucleic acid, it can To test single-stranded double-helix DNA, or to analyze triple-helix formation, the hybridization ability depends on the degree of complementarity of the antisense nucleic acid and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with Neutrokine-α and/or Neutrokine-αSV RNA, which can contain and still form a stable double helix (or triple helix). The permissible degree of mismatching can be ascertained by one skilled in the art using standard methods to determine the melting point of the hybridization complex.

Oligonucleotides complementary to the 5' end of the message, such as the 5' untranslated sequence close to and including the AUG initiation codon, should inhibit translation more effectively. However, sequences complementary to the 3' untranslated sequences of mRNA have been shown to effectively inhibit translation of mRNA. See Wagner, R. 1994, Nature 372:333-335. Therefore, oligonucleotides shown in Figures 1A-B and 5A-B that are complementary to the 5' or 3' untranslated non-translated regions of Neutrokine-α and/or Neutrokine-αSV, respectively, can be used in antisense methods to Inhibits translation of endogenous Neutrokine-α and/or Neutrokine-αSV mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the coding region of mRNA are minor translational inhibitors, but may be used in accordance with the present invention. Regardless of whether it hybridizes to the 5', 3' or coding region of Neutrokine-α and/or Neutrokine-αSV mRNA, the length of the antisense nucleic acid should be 6 nucleotides, preferably 6-50 nucleotides. In particular, the oligonucleotide is at least 10, 17, 25 or at least 50 nucleotides in length.

The polynucleotides of the invention may be DNA or RNA or chimeric mixtures or derivatives or modified forms thereof, and may be single-stranded or double-stranded. Oligonucleotides may be modified in base moiety, sugar moiety, or phosphate backbone, eg, to improve molecular stability, hybridization, and the like. The oligonucleotide may include other appended groups such as peptides (e.g., to target host cell receptors in vivo), or agents to facilitate transmembrane transport (see, e.g., Letsinger et al., 1989, Proc. Lemaitre et al., Proceedings of the National Academy of Sciences 84:648-652 (1987); PCT Publication No. WO88/09810, published December 15, 1988) or preparations that cross the blood-brain barrier (see, e.g., PCT Publication WO89/10134, Published April 25, 1988), hybridization-initiating cleavage agents (see, eg, Krol et al., Biotechnology 6:958-976 (1988)) or intercalators (see, eg, Zon, Pharmaceutical Research 5:539-549 (1988)) . Thus, an oligonucleotide can be conjugated to another molecule, such as a peptide, a hybridization-initiating crosslinker, a transport agent, a hybridization-initiating cleavage agent, and the like.

Antisense oligonucleotides may comprise a modified base moiety selected from the group consisting of, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, yellow Purine, 4-acetylcytosine, 5-carboxymethyluracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D -Galactose queosine, inosine, N6-prenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine Baseguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thio Uracil, β-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-prenyl adenine, uracil-5-oxyacetic acid (v), Wybutoxiosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxo Methyl acetate, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-carboxypropyl)uracil, (acp3)w, and 2,6-Diaminopurine.

Antisense oligonucleotides may also comprise at least one modified sugar component selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose and hexose.

In another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, phosphorodithioate, phosphorothioate, phosphonothioate, phosphoramidite, Phosphodiamide, methyl phosphate, alkyl phosphate triester, formacetal or their analogs.

In another embodiment, the antisense oligonucleotide is an alpha-anomeric oligonucleotide. Alpha-terminal oligonucleotides form specific double-stranded hybrids with complementary RNA in which, contrary to typical beta units, the strands run parallel to each other (Gautier et al., Nucleic Acids Res. 15:6625-6641 (1987)). Oligonucleotides are 2-O-methylribonucleotides (Inoue et al., Nucleic Acids Res. 15:6131-6148 (1987)), or chimeric RNA-DNA analogs (Inoue et al., FEBS Communications 215:327-330 (1997)).

The polynucleotides of the present invention can be synthesized by standard methods known in the art, for example using an automatic DNA synthesizer (such as commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioates can be synthesized by the method of Stein et al. (Nucleic Acids Res. 16:3209 (1988)), and methylphosphonate oligonucleotides can be prepared by controlled pore glass polymer supports (Sarin et al., Proc. 85: 7448-7451 (1988)) et al.

Antisense nucleotides complementary to the sequence of the Neutrokine-[alpha] and/or Neutrokine-[alpha]SV coding region may be used, preferably antisense nucleotides complementary to the transcribed untranslated region.

Potential antagonists of the present invention also include catalytic RNA or ribozymes (see, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., Science 247:1222-1225 (1990). Can be used at sites A ribozyme that specifically recognizes a sequence that cleaves mRNA to destroy Neutrokine-α and/or Neutrokine-αSV mRNA, preferably using a hammerhead ribozyme. Hammerhead ribozyme cleavage is located on both sides of the region that forms complementary base pairs with the target mRNA The only requirement is that the target mRNA has a 5'-UG-3' sequence. The construction and production of hammerhead ribozymes are well known in the art and are described in detail in Haseloff and Gerlach, Nature 334:585-591 (1988) Described. There are many potential hammerhead ribozyme cleavage sites (see respectively in Fig. 1A-B and 5A-B) in the nucleotide sequence of Neutrokine-α and/or Neutrokine-αSV.Preferably, this ribozyme is carried out Engineered so that the cleavage recognition site is located near the 5' end of the Neutrokine-α and/or Neutrokine-αSV mRNA, ie to increase potency and minimize intracellular accumulation of non-functional mRNA transcripts.

In the antisense approach, the ribozymes of the invention may consist of modified oligonucleotides (eg improved stability, orientation, etc.) and should be delivered to cells expressing Neutrokine-α and/or Neutrokine-αSV in vivo. A DNA construct encoding a ribozyme can be introduced into cells in the same manner as described above to introduce antisense encoding DNA. Preferred methods of delivery involve the use of DNA constructs that "encode" ribozymes under the control of a strong constitutive promoter, such as the polIII, or polII promoter, so that transfected cells will produce sufficient ribozymes to destroy endogenous Neutrokine-α and/or Neutrokine-αSV messenger, and inhibits translation. Since ribozymes are not catalytic like antisense molecules, lower intracellular concentrations are required to increase potency.

Endogenous gene expression can also be reduced by inactivating or "knocking out" the Neutrokine-α and/or Neutrokine-αSV genes and/or their promoters using directed homologous recombination (e.g. Smithies et al., Nature 317:230-24( 1985); Thomas and Capeahi, Cell 51:503-512 (1987); Thompson et al, Cell 5:313:321 (1989); all of which are incorporated by reference in their entirety). For example, a mutated non-functional polynucleotide of the invention (or a complementary unrelated DNA sequence) flanked by DNA (coding or regulatory region of a gene) homologous to an endogenous polynucleotide sequence may be used, It can be used with or without selectable markers and/or negative selectable markers to transfect cells expressing the polypeptides of the invention in vivo. In another embodiment, a knockout is generated in a cell that contains but does not express the corresponding gene using methods known in the art. The DNA construct is inserted by directed homologous recombination, resulting in directed gene inactivation. This method is particularly suitable for research and agricultural fields, where modification of embryonic stem cells can be used to produce animal progeny with inactivated directed genes (see Table Thomas and Capechi 1987 and Thompson 1989 supra). However, this method can be routinely adapted to humans, provided the recombinant DNA construct is administered or directed to the desired site in vivo using appropriate viral vectors, as will be apparent to those skilled in the art. All cited documents are incorporated by reference in their entirety.

In other embodiments, antagonists of the invention include soluble forms of Neutrokine-α and/or Neutrokine-αSV (fragments of Neutrokine-α as shown in Figure 1A-B, including ligand binding domain, TNF conserved The domain of Neutrokine-α and/or the extracellular domain of Neutrokine-αSV, and the fragment of Neutrokine-αSV shown in Figure 5A-B, including ligand binding domain, TNF conserved domain, and/or or Neutrokine-α and/or the extracellular domain of Neutrokine-αSV). This soluble form of Neutrokine-α and/or Neutrokine-αSV, which may be naturally occurring or synthetic, binds Neutrokine-α and/or Neutrokine-αSV by competing with native Neutrokine-α and/or Neutrokine-αSV. body (such as DR5 (see International Publication WO98/41629), TR10 (see International Publication WO98/54202), 312C2 (see International Publication WO98/06842) and TR11, TR11SV1 and TR11SV2 (see U.S. Patent No. 09/176200)), Or antagonize Neutrokine-α and/or Neutrokine-αSV-mediated signaling by forming multimers that can bind to receptors or not, but cannot induce signal transduction. Preferably, these antagonists inhibit Neutrokine-[alpha] and/or Neutrokine-[alpha]SV-mediated stimulation of lymphocyte (eg B cell) proliferation, differentiation and/or activation. The antagonists of the present invention also include antibodies specific to TNF family ligands (such as CD30) and Neutrokine-α-Fc and/or Neutrokine-αSV-Fc fusion proteins.

"TNF family ligands" refers to naturally occurring, recombinant and synthetic ligands that bind TNF receptor family members and induce and/or block ligand/receptor signaling pathways. Members of the TNF ligand family include, but are not limited to, TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (in the heterotrimeric LT-α-2-β complex discovered), FasL, CD40L, (TNF-γ (International Publication WO96/14328), ATIM-I (International Publication WO97/33899), AIM-II (International Publication WO97/34911), APRIL (J.Exp.Med. 188(6):1185-1190), α-intrinsic factor (International Publication WO98/07880), α-Neurofactor (International Publication WO97/18921), CD27L, CD30L, 4-1BBL, OX40L, CD27, CD30, 4- 1BB, OX40 and nerve growth factor (NGF).In a preferred embodiment, Neutrokine-α and/or Neutrokine-αSV TNF family ligand of the present invention is DD5 (seeing International Publication WO98/41629), TR10 (seeing International Publication WO98/54202), 312C2 (see International Publication WO98/06842) and TR11, TR11SV1 and TR11SV2 (see US Patent Application No. 09/176200).

Antagonists of the invention also include antibodies specific to TNF family receptors or Neutrokine-α and/or Neutrokine-αSV polypeptides of the invention. Antibodies of the invention can be prepared using the Neutrokine-α and/or Neutrokine-αSV immunogens of the invention by various standard methods. This Neutrokine-alpha and/or Neutrokine-alpha SV immunogen comprises complete Neutrokine-alpha and/or Neutrokine shown in Figure 1A-B (SEQ ID NO:2) and Figure 5A-B (SEQ ID NO:19), respectively - αSV polypeptides (which may or may not include a leader sequence), and Neutrokine-α and/or Neutrokine-αSV polypeptide fragments comprising, for example, a ligand binding domain, a TNF conserved domain, an extracellular domain, a transmembrane domain, and /or an intracellular domain, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists of the present invention can be obtained according to Tartaglia and Goeddel, Journal of Biochemistry 267 (7): 4304-4307 (1992); Tartaglia et al., Cell 73: 213-216 (1993) and PCT The method shown in application WO94/09137 produces, and preferably is specific to (ie only binds to) the polypeptide of the invention having the amino acid sequence of SEQ ID NO:2. The term "antibody" (Ab) or "monoclonal antibody" (mAb) is meant to include whole molecules capable of binding antigen as well as fragments thereof) such as Fb and F(ab') fragments). Fab, Fab' and F(ab') fragments lack the Fc fragment intact antibody, which is more rapidly cleared from circulation and may have less nonspecific tissue binding of intact antibody (Wahl et al., J Nucleic Acid Methods 24:316-325 (1983)).

In a preferred method, the antibodies of the invention are mAbs. Such mAbs can be prepared by the hybridoma method (Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Harlow et al., Antibody Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: New Advances in Biological Analysis, Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody Technology," Experimental Methods in Biochemistry and Molecular Biology, Vol. 13 (Burdor et al., Elsevier, Amsterdam (1984)).

Proteins and other compounds that bind Neutrokine-[alpha] and/or Neutrokine-[alpha]SV domains are also candidate agonists and antagonists of the invention. Such binding compounds can be "captured" using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)). The yeast two-hybrid system has been modified by Roger Brent and colleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell 72:223-232 (1993)). Preferably, compounds that bind to the ligand-binding domain, extracellular domain, intracellular domain, transmembrane domain, and death domain of Neutrokine-α and/or Neutrokine-αSV are captured using the yeast two-hybrid system according to the present invention. Such compounds are good candidates for agonists and antagonists of the invention.

For example, using the two-hybrid assay described above, the ectodomain and intracellular domain of Neutrokine-α and/or Neutrokine-αSV receptors, or a portion thereof, can be used to identify interactions with Neutrokine-α and/or Neutrokine-αSV receptors in vivo of cellular proteins. This analysis can also be used to identify cellular proteins that interact with Neutrokine-[alpha] and/or Neutrokine-[alpha]SV receptors in vivo. This assay can also be used to identify ligands with potential agonistic or antagonistic activity for Neutrokine-[alpha] and/or Neutrokine-[alpha]SV receptor function. This screening assay was previously used to identify proteins that interact with the cytoplasmic domain of mouse TNF-RII and to identify two receptor-associated proteins. Rothe et al., Cell 78:681 (1994). Such proteins and amino acid sequences that bind the cytoplasmic region of Neutrokine-alpha and/or Neutrokine-alpha SV receptors are good candidates for agonists and antagonists of the invention.

Other screening methods include the use of cells expressing a polypeptide of the invention (eg, transfected CHO cells) to measure changes in extracellular pH resulting from receptor activation, as described in Science 246:181-296 (1989). In another example, a potential agonist or antagonist can be contacted with cells expressing a polypeptide of the invention, and a second messenger response, such as signal transduction, can be assayed to determine whether the potential antagonist or agonist is effective.

Agonists of the present invention include naturally occurring and synthetic compounds such as TNF family ligand peptide fragments, transforming growth factors, neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate), Tumor suppressor (p53), cytolytic T cells and antimetabolites. Preferred agonists include chemotherapeutic drugs and cisplatin, doxorubicin, bleomycin, cytarabine, nitrogen mustard, methotrexate and vincristine. Other agonists include ethanol and starch peptides (Science 267:1457-1458 (1995)).

Preferred agonists are Neutrokine-[alpha] and/or Neutrokine-[alpha]SV fragments of the invention which stimulate lymphocyte (eg B cell) proliferation, differentiation and/or activation. Further preferred agonists include polyclonal and monoclonal antibodies raised against Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptides of the invention, or fragments thereof. Such agonist antibodies raised against TNF family receptors are described in Tartaglia et al., PNAS USA 88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem. 267:4304-4307 (1992). See also described in PCT application WO 94/09137.

In another embodiment, immunomodulatory molecules such as IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-γ and TNF-α, can be used as the Neutrokine of the present invention. - an agonist of alpha and/or Neutrokine-alpha SV polypeptide which stimulates lymphocyte (eg B cell) proliferation, differentiation and/or activation. In a specific embodiment, IL4 and/or IL10 is used to proliferate Neutrokine-[alpha] and/or Neutrokine-[alpha]SV-mediated B cell proliferation.

In another embodiment of the present invention, cells engineered to express a polypeptide of the present invention, or cells engineered not to express a polypeptide of the present invention (eg, knockout), are administered to a patient in vivo. Such cells may be obtained from a patient (i.e., animal, including human), or an MHC compatible donor, and may include, but are not limited to, fibroblasts, bone marrow cells, blood cells (such as lymphocytes), adipocytes, muscle cells, endothelial cells wait. Cells are genetically engineered in vitro using recombinant DNA methods to introduce the coding sequence for the polypeptide of the present invention into the cell, or to disrupt the coding sequence and/or endogenous regulatory sequences associated with the polypeptide of the present invention, such as by transduction (with a viral vector , and preferably a vector for integrating the transgene into the cell genome), or transfection methods, including but not limited to, using plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. A coding sequence for a polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or a promoter/proliferator for the expression, preferably secretion, of the polypeptide of the invention. Genetically engineered cells expressing and preferably secreting a polypeptide of the invention can be introduced into the patient systemically, eg, by circulation or intraperitoneal injection.

Alternatively, cells can be incorporated into a matrix and implanted in vivo, such as genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft (See, eg, Anderson et al., US Patent No. 5,399,349; and Mulligan and Wilson, US Patent No. 5,460,959, both incorporated by reference in their entirety).

When the administered cells are non-self or non-MHC compatible cells, they can be administered by well-known methods to prevent the host from mounting an immune response to the introduced cells. For example, cells can be introduced in capsule form, allowing immediate exchange of components into the extracellular environment, but without allowing the introduced cells to be recognized by the host's immune system.

In another embodiment of the invention, the activity of Neutrokine-α and/or Neutrokine-αSV polypeptides may be reduced by "dominant negative". For this reason, constructs encoding defective Neutrokine-α and/or Neutrokine-αSV polypeptides such as mutants that lack all or part of the TNF conserved domain can be used in gene therapy to weaken Neutrokine-α and/or Neutrokine-αSV on activity of appropriate target cells. For example, nucleotide sequences directing host cells to express Neutrokine-α and/or Neutrokine-αSV polypeptides can be introduced into monocytes or other cells or tissues (by in vivo or ex vivo gene therapy or other methods known in the art) ), all or part of the conserved domains of TNF in the nucleotide sequence have been changed or deleted. Alternatively, directed homologous recombination can be used to introduce such deletions or mutants into the endogenous Neutrokine-[alpha] and/or Neutrokine-[alpha]SV genes in the monocytes of the subject individual. The engineered cells will express non-functional Neutrokine-α and/or Neutrokine-αSV polypeptides (ie, ligands (eg, multimers) that bind but cannot induce signal transduction).

chromosome analysis

The nucleic acid molecules of the invention can also be used for chromosome identification. This sequence is specifically oriented and capable of hybridizing to a particular location on an individual human chromosome. In addition, it is often necessary to identify specific loci on chromosomes. Few chromosomal labeling reagents based on real sequence data (repeat polymorphisms) are suitable for labeling chromosomal locations. Chromosomal DNA mapping of the present invention is an important first step in determining the association of these sequences with disease-associated genes.

In some preferred embodiments, the cDNA and/or polynucleotides set forth herein are used to clone the genomic DNA of the Neutrokine-α and/or Neutrokine-αSV gene. This can be done using various well known methods and commercially available libraries. This genomic DNA is then used for in situ chromosome mapping using well known methods.

Additionally, in some cases, the sequence can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp) from the cDNA. In silico analysis of the 3' untranslated region of a gene for the rapid selection of primers that span no more than one exon within the genome and otherwise complicate amplification. These primers were then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Fluorescence in situ hybridization (FISH) of cDNA clones on metaphase chromosomal smears can be used to provide precise chromosomal localization in one step. This method can use probes as short as 50 or 60 bp from cDNA. This method is described in Verma et al., A Handbook of Basic Human Chromosome Technique, Pergamon Press, New York (1988).

Once the sequence is pinpointed on the chromosome, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data are found, eg, in V McKusick, Mendelian Inheritance In Man, available online from Johns Hopkins University, Welch Medical Library. Relationships between genes and diseases that have been mapped to the same chromosomal region are then identified by pedigree analysis (naturally in joint genetic signatures of adjacent genes).

The next step is to determine the differences in cDNA or genome sequences between affected and non-affected individuals. A mutation is responsible for a disease if it is observed in some or all affected individuals but not in normal individuals.

The precise positioning of cDNA on the chromosome is related to diseases caused by 50 to 500 potential pathogenic genes through the identification of physical maps and genetic maps. (assuming 1 megabase map resolution, and one gene per 20 kb).

Using the method described above, the chromosomal location of Neutrokine-α and/or Neutrokine-αSV can be determined with high confidence on chromosome 13g34 using a combination of somatic hybrids and radiation hybrids.

Example

The present invention will be readily understood by reference to the following examples, which illustrate and do not limit the invention. Many of the following examples specifically illustrate the Neutrokine-alpha polynucleotides and polypeptides of the invention. Each of the examples is also applicable to the production and/or detection of Neutrokine-αSV polynucleotides and polypeptides of the present invention. Those skilled in the art can easily carry out the examples about Neutrokine-αSV through the following examples.

Example 1a: Expression and purification of "His-tagged" Neutrokine-α in E. coli

Bacterial expression was performed using the bacterial expression vector pQE9 (pD10) in this example. (QIAGEN Corporation, supra). pQE9 encodes ampicillin resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS") encoding a histidine residue 6 codons, and an appropriate single restriction enzyme cleavage site, the histidine residues can be affinity purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity resin sold by QIAGEN. These factors are arranged such that the inserted DNA segment encoding a polypeptide encodes a polypeptide having six histidine residues (ie, a 6X His tag) covalently linked to the amino terminus of the polypeptide.

The DNA sequence encoding the desired portion of the Neutrokine-α protein containing the ectodomain sequence was amplified from a deposited cDNA clone using PCR oligonucleotide primers that annealed individually to the amino-terminal sequence of the desired portion of the protein , and 3' of the cDNA coding sequence in the deposited construct sequence. Additional nucleotides containing restriction sites in the pQE9 vector to facilitate cloning were added to the 5' and 3' primer sequences, respectively.

To clone the extracellular domain of the protein, the sequence of the 5' primer is 5'-GTG GGA TCC A GC CTC CGG GCA GAG CTG-3' (SEQ ID NO: 10), which contains the underlined Bam HI restriction site, followed by Figure 18 nucleotides of the N-terminal coding sequence of the ectodomain of the sequences shown in 1A and 1B. Of course, those skilled in the art know that the position at which the 5' primer starts within the protein coding sequence can be varied to amplify a DNA segment encoding any desired portion of the complete Neutrokine-alpha protein, which is longer than the extracellular domain or short. The sequence of the 3' primer is 5'-GTG AAG CTT TTA TTACAG CAG TTT CAA TGC ACC-3' (SEQ ID NO: 11), which contains an underlined Hind III restriction site followed by 2 stop codons, and complementary 18 nucleotides of the coding sequence at the 3' end of the DNA sequence shown in Figures 1A and 1B.

The amplified DNA fragment and the vector pQE9 were digested with Bam HI and Hind III, and then the digested DNAs were ligated together. DNA inserted into the restricted pQE9 vector placed the protein coding region downstream of the IPTG inducible promoter and in frame with the initial AUG and six histidine codons.

The ligation mixture is transformed into appropriate E. coli cells by standard methods as described by Sambrook et al., A Laboratory Manual of Molecular Cloning, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). E. coli strain M15/rep4 containing multiple copies of plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance (Kan ^r ), was used to perform the examples illustrated herein. This strain is only one of many strains suitable for protein expression, which are commercially available from the company QIAGEN. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA was isolated from resistant clones and cloned DNA was confirmed by restriction analysis, PCR and DNA sequencing. Clones containing the desired construct were grown overnight (O/N) in liquid LB medium supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). This O/N culture was used to inoculate a larger culture at a dilution of approximately 1:25 to 1:250. Cells were grown to an optical density at 600 nm (OD600) between 0.4-0.6.

IPTG was then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter by inactivating the lac repressor. Cells were then incubated for an additional 3-4 hours. Cells were then collected by centrifugation.

Cells were then agitated in 6M guanidine hydrochloride, pH 8 at 4°C for 3-4 hours. Cell debris was removed by centrifugation, and the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) affinity resin chromatography column (purchased from QIAGEN). Proteins with 6×His tags bind to Ni-NTA resin with high affinity and can be purified by a simple one-step method (see QIAexpressionist, 1995, QIAGEN Company for details). Briefly, the supernatant was loaded onto a chromatography column in 6M guanidine hydrochloride, pH 8, the column was first washed with 10 volumes of 6M guanidine hydrochloride, pH 6, and finally Neutokine-α was eluted with 6M guanidine hydrochloride, pH 5 and Neutokine-αSV polypeptide.

The purified protein was then refolded by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while being immobilized on a Ni-NTA column. The applicable conditions are as follows: in 500mM NaCl containing protease inhibitors, 20% glycerol, 20mM Tris/HCl pH7.4, renaturation with a linear 6M-1M urea gradient. Renaturation should be carried out for 1.5 hours or more. After renaturation, the protein can be eluted by adding 250 mM imidazole. Imidazole was removed by final dialysis against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. Store purified protein at 4°C or freeze at -80°C.

Example 1b: Expression and purification of Neutokine-αSV in E. coli

In this example bacterial expression was performed using the bacterial expression vector pQE60. (QIAGEN Corporation, 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS") encoding a histidine residue 6 codons, and an appropriate single restriction enzyme cleavage site, the histidine residues can be affinity purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity resin sold by QIAGEN. These elements are arranged such that the inserted DNA fragment encoding the polypeptide encodes a polypeptide having 6 histidine residues (i.e. 6X His tag), and these 6 histidine residues are covalently linked to the amino terminus of the polypeptide. However, in this example, a polypeptide coding sequence is inserted, thereby preventing translation of the 6-histidine codon, and thus producing a polypeptide without the 6-histidine tag.

The DNA sequence encoding the desired portion of the protein comprising the extracellular domain sequence was amplified from the deposited cDNA clone using PCR oligonucleotide primers that annealed to the amino-terminal sequence of the desired portion of the protein, respectively, and the deposited 3' of the cDNA coding sequence in the sequence of the construct. Additional nucleotides containing restriction sites in the pQE9 vector to facilitate cloning were added to the 5' and 3' primer sequences, respectively.

To clone the extracellular domain of the protein, the 5' primer had the sequence 5'-GTG TCA TGA GCC TCC GGG CAG AGC TG-3' (SEQ ID NO: 12), which contained the underlined Bsp HI restriction site, followed by Figure 1A and 17 nucleotides of the N-terminal coding sequence of the ectodomain of the sequence shown in 1B. Of course, those skilled in the art will appreciate that the position at which the 5' primer starts within the protein coding sequence can be varied to amplify any desired portion of the encoded complete protein, which is longer or shorter than the extracellular domain. The sequence of the 3' primer is 5'-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID NO: 13), which contains an underlined Hind III restriction site followed by 2 stop codons, and 18 nucleotides complementary to the 3' coding sequence of the DNA sequence shown in Figures 1A and 1B.

The amplified DNA fragment and the vector pQE60 were digested with Bsp HI and Hind III, and then the digested DNAs were ligated together. DNA inserted into the restricted pQE60 vector places the protein coding region downstream of the IPTG inducible promoter and in frame with the initial AUG and six histidine codons. An associated stop codon prevents translation of the 6 histidine codons downstream of the insertion point.

The ligation mixture is transformed into appropriate E. coli cells by standard methods as described by Sambrook et al., A Laboratory Manual of Molecular Cloning, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). E. coli strain M15/rep4 containing multiple copies of plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance (Kan ^r ), was used to perform the examples illustrated herein. This strain is one of many strains suitable for expressing proteins and is commercially available from QIAGEN Corporation. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA was isolated from resistant clones and cloned DNA was confirmed by restriction analysis, PCR and DNA sequencing.

Those skilled in the art recognize that any of a number of bacterial expression vectors can be used in place of pQE9 and pQE60 used in the expression method of this example. For example, the new pHE4 series of bacterial expression vectors, particularly the pHE4-5 vector, can be used for bacterial expression in this example (ATCC No. 209311; and variants thereof). A plasmid DNA called pHE4-5/MPIFD23, ATCC Accession No. 209311, is a vector plasmid DNA containing an insert encoding another ORF. This construct was deposited on September 30, 1997 with the American Type Culture Collection, located at 20110-2209 University Drive, Manassas, VA 10801. Using the Nde I and Asp 718 restriction sites flanking the irrelevant MPIF ORF insert, one skilled in the art can readily use current molecular biology methods to replace the irrelevant pHE4-5 vector with the Neutrokine-α ORF of the present invention. ORF.

The pHE4-5 bacterial expression vector includes the neomycin phosphotransferase gene for selection, an E. coli origin of replication, a T5 phage promoter sequence, two lac operator sequences, a Shine-Delgarno sequence, and a lactose operon repressor The biological gene (lacIq). The arrangement of these elements is such that the DNA fragment encoding the polypeptide is inserted to express a polypeptide with 6 histidine residues (ie 6×His tag), and the 6 histidine residues are covalently linked to the amino terminal of the polypeptide. The promoter and operator sequences of the pHE4-5 vector were produced synthetically. Methods for the synthetic production of nucleic acid sequences are well known in the art (CLONETECH 95/96 Catalog, p215-216, CLONETECH, 1020 East Meadow Circle, Palo Alto, CA94303).

Clones containing the desired Neutokine-αSV construct were grown overnight (O/N) in liquid LB medium supplemented with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). This O/N culture was used to inoculate a larger culture at a dilution of approximately 1:25 to 1:250. Cells were grown to an optical density at 600 nm (OD600) between 0.4-0.6. IPTG was then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter by inactivating the lac repressor. Cells were then incubated for an additional 3-4 hours. Cells were then collected by centrifugation.

Cells were then agitated in 6M guanidine hydrochloride, pH 8 at 4°C for 3-4 hours. Cell debris was removed by centrifugation, and the supernatant containing Neutrokine-α was dialyzed against 50 mM sodium acetate pH 6 buffer supplemented with 200 mM NaCl. Alternatively, the protein can be successfully refolded by dialysis against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4 containing protease inhibitors. After renaturation, proteins can be purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. Alternatively, affinity chromatography such as antibody chromatography columns can be used to obtain purified proteins. Store purified protein at 4°C or freeze at -80°C.

In some embodiments, it is preferred to generate expression constructs as described in this example to mutate one or more of the 3 cysteine residues in the Neutrokine-alpha polypeptide sequence. The cysteine residues in the Neutrokine-α polypeptide sequence are located at positions 147, 232, and 245 shown in SEQ ID NO: 2, and at positions 213 and 226 shown in SEQ ID NO: 19 (Neutrokine-α SV polypeptide sequence There is no cysteine corresponding to Cys-147 in the Neutrokine-α polypeptide sequence because amino acid residues 143-160 of the Neutrokine-α polypeptide sequence do not exist in the Neutrokine-αSV polypeptide sequence).

Example 2: Cloning, expression, and purification of Neutrokine-α protein in the baculovirus expression system

In this example, the plasmid shuttle vector pA2GP was used to insert the cloned DNA encoding the protein ectodomain, deleted of its naturally associated intracellular and transmembrane sequences, into baculovirus to express the ectodomain of the Neutrokine-α protein, A baculovirus leader sequence and a method as described in Summers et al., Baculovirus and Insect Cell Culture Methods Manual, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) were used. This expression vector contains the strong polyhedrin promoter of AcMNPV, followed by the secretion signal peptide (leader sequence) of the baculovirus gp67 protein, and conventional restriction sites such as BamHI, Xba I and Asp 718 restriction sites. The polyadenylation site of Simian Virus 40 (SV40) is used for efficient polyadenylation. For easy selection of recombinant viruses, the plasmid contains the β-galactosidase gene from E. coli under the control of a weak Drosophila promoter in the same direction, followed by the polyadenylation signal for the polyhedrin gene. The inserted genes flank the viral sequence to allow cell-mediated homologous recombination with wild-type viral DNA to generate live virus expressing the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the above-mentioned vectors, such as pAc373, pVL941, and pAcIM1, as long as those skilled in the art realize that this construct provides the transcription, translation, secretion, etc. signals in appropriate positions, including the required signal peptide and AUG in the box. Such vectors are described, for example, in Luckow et al., Virology 170:31-39 (1989).

The deposited clone encodes the ectodomain of Neutrokine-α protein in N-terminal deleted form, lacks the AUG start codon, naturally associated intracellular and transmembrane domain sequences, and is shown in Figures 1A and 1B (SEQ ID NO: 2) The cDNA sequence of amino acids Gln-73 to Leu-79 was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. The 5' primer sequence is 5'-GTG GGA TCC CCG GGC AGA GCTGCA GGG C-3' (SEQ ID NO: 14), which contains the underlined Bam HI restriction enzyme site followed by Neutrokine-α shown in Figures 1A and 1B Eighteen nucleotides of the protein ectodomain sequence, starting at the N-terminus of the indicated protein ectodomain. The 3' primer sequence is 5'-GTG GGA TCC TTA TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID NO: 15), which contains an underlined Bam HI restriction enzyme site followed by two stop codons, and Complementary to 18 nucleotides of the 3' coding sequence shown in Figures 1A and 1B.

In some other embodiments, constructs expressing the entire putative extracellular domain of Neutrokine-alpha (ie amino acid residues Gln-73 to Leu-285) are preferred. Those skilled in the art can use the polynucleotide and polypeptide sequences provided by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, to design polynucleotide primers to generate such clones.

In a preferred embodiment, the pA2GP expression construct encodes amino acid residues from Leu-112 to Leu-285 of the Neutrokine-α polypeptide sequence shown in SEQ ID NO:2.

In another preferred embodiment, the pA2GP expression construct encodes amino acid residues from Ser-78 to Leu-285 of the Neutrokine-α polypeptide sequence shown in SEQ ID NO:2.

The amplified fragments were separated from 1% agarose gel using a commercially available kit (Geneclean, BIO 101 Company, La Jolla, Ca.). This fragment was then digested with BamHI and further purified on a 1% agarose gel. This fragment is referred to herein as F1.

The plasmid was digested with the restriction enzyme BamHI and optionally dephosphorylated with calf intestinal phosphatase using conventional methods known in the art. DNA was then separated from 1% agarose gel using a commercially available kit (Geneclean, BIO 101 Company, La Jolla, Ca.). This vector DNA is referred to herein as V1.

Fragment F1 and dephosphorylated plasmid V1 were ligated with T4 DNA ligase. Escherichia coli HB101 or other suitable Escherichia coli hosts such as XL-1Blue cells (Statagene Cloning Systems, La Jolla, Ca.) were transformed with this ligation mixture, and spread on culture plates. Bacteria containing plasmids with human genes were identified by digesting the DNA of individual colonies with BamHI, and then analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragments was determined by DNA sequencing. This plasmid is referred to herein as pA2GP-Neutrokine-alpha.

5 μg of plasmid pA2GP-Neutrokine-α was co-transfected with 1.0 μg of commercially available linear baculovirus DNA (BaculoGold ^™ Baculovirus DNA, Pharmingen, San Diego, CA) using, for example, Felgner et al., Proc. 7413-7417 (1987) described lipostaining method. 1.0 μg of BaculoGold ^™ viral DNA was mixed with 5 μg of plasmid pA2GP-Neutrokine-α in sterile wells of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies, Inc., Gaithersburg, MD). Afterwards, 10 μl of Lipofectin and 90 μl of Grace's medium were added, mixed and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL 1711 ), which were seeded in 35 mm tissue culture plates containing 1 ml of serum-free Grace's medium. The plates were then incubated at 27°C for 5 hours. The transfection solution was then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% calf serum was added. Then culture was continued for 4 days at 27°C.

After 4 days, supernatants were collected and subjected to plaque analysis as described by Summers and Smith. Gal-expressing clones producing blue-stained plaques were easily identified and isolated using agarose gels with "Blue Gal" (Life Technologies, Inc., Rockville, Maryland). (A detailed description of this type of plaque assay can also be found in Life Technologies, Inc., Rockville, Maryland, Insect Cell Culture and Baculovirus Distribution Instructions, pages 9-10). After appropriate incubation, blue-stained plaques are picked with the tip of a micropipette (eg, Eppendorf). The agar containing the recombinant virus was then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium, and the suspension containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants from these culture plates were collected and stored at 4°C. This recombinant virus is called V-Neutrokine-alpha.

To examine the expression of the Neutrokine-α gene, Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-Neutrokine-[alpha] at a multiplicity of infection (MOI) of approximately 2. If radiolabeled proteins were desired, the medium was removed after 6 hours and replaced with SF900II medium (purchased from Life Technologies, Inc., Rockville, Maryland) minus methionine and cysteine. After 42 hours, 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S-cysteine (purchased from Amersham) were added. Cells were harvested by centrifugation after incubation for 16 hours. Proteins in the supernatant, as well as intracellular proteins, were analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence at the N-terminus of purified proteins can be used to determine the N-terminal sequence of the extracellular domain of the protein, and thus determine the cleavage site and the length of the secretion signal peptide.

In a specific embodiment, recombinant Neutrokine-α was purified from the supernatant of baculovirus-infected Sf9 cells, as described below. Insect cells were grown in EXCEL401 medium (JRH Scientific) with 1% (v/v) calf serum. After 92 hours of infection, the collected supernatant was clarified by centrifugation at 18000 xg followed by filtration at a depth of 0.45 m. A skim filtration step can also be used to remove lipid contaminants and subsequently improve the initial capture of Neutrokine-alpha protein.

Supernatants were randomly loaded into a series of Poros HS-50/H-50. Alternatively, Toyopearl QAE, Toyopearl Super Q (Tosohass), Q-Sepharose (Pharmacia) and equivalent resins can be used. This step is used as a negative purification step to remove strong anion-binding contaminants. The HS/HQ flowing through the raw material was adjusted to pH 7.5 with 1M Tris-HCl pH 8, diluted with an equal volume of 50mM Tris-HCl pH 8, and loaded on a porous PI-20 or PI-50 chromatography column. The PI chromatography column was first washed with 4 volumes of 75mM NaCl in 50mM Tris-HCl pH7.5, and then eluted with 3-5 column volumes of 300mM, 750mM, 1500mM NaCl in 50mM Tris-HCl pH7.5. Neutrokine-α protein presents a 17KD band on simplified SDS-PAGE, and exists in the 0.75M-1.5M NaCl fraction.

The PI fraction was further purified through a Sephacryl S100HR (Pharmacia) size exclusion column equilibrated with 0.15M NaCl, 50mM sodium acetate, pH6. The S200 fraction was mixed with NaCl to a final concentration of 3M, and loaded on a Toyopearl Hexyl 650C (Tosohass) chromatographic column. The Hexyl column was eluted with 5-10 volumes of a linear gradient of 3M-0.05M NaCl in 50 mM sodium acetate pH6. The NaCl gradient in Hexyl chromatography can also be replaced by a 1M-0M gradient of ammonium sulfate in 50mM sodium acetate pH6. Fractions containing Neutrokine-α purified by SDS-PAGE analysis were combined and dialyzed against a solution containing 150 mM NaCl, 50 mM sodium acetate, pH 6.

The final purified Neutrokine-alpha protein, expressed in the baculovirus system described herein, has an N-terminal sequence from the Ala-134 amino acid residue of SEQ ID NO:2. RP-HPLC analysis showed a single peak above 95% purity. Endotoxin levels were below the detection limit of the LAL assay.

In another example, recombinant Neutrokine-α was purified from baculovirus-infected Sf9 cells containing 0.25% bovine serum, as described below.

Sf9 supernatant was collected by centrifugation at 18000 xg. Then the supernatant was treated with 10 mM CaCl ₂ under mild alkaline conditions for 10-15 minutes, followed by centrifugation and 0.22 μm depth filtration. Then the resulting Sf9 cell supernatant was diluted 2-fold, and loaded on a Poros PI-50 chromatography column (purchased from PE Biosystems). The column was equilibrated with 50 mM Tris (pH=7.4). The PI-50 column was washed with 1 CV of 50 mM Tris (pH=7.4) and then eluted with 3 CV+ of 1.5M NaCl in 50 mM NaOAc (pH=6). The PI fraction was loaded onto a Sephacryl S200 chromatography column equilibrated with 50 mM NaOAc (pH=6), 125 mM NaCl. The S200 fraction was mixed with salt to a final concentration of 0.7M ammonium sulfate and 0.6M NaCl, and loaded on a Toyopearl Hexyl 650C chromatography column (purchased from Toso Haas), which had been used to contain 50mM NaOAc (pH=6) Equilibrate in 0.6M NaCl, 0.7M ammonium sulfate buffer. The column was then rinsed with 2CV of the same buffer. Recombinant Neutrokine-α was then eluted with 3CV of 50mM NaOAc (pH=6), followed by 2CV of 20% ethanol. The recombinant Neutrokine-alpha protein was then eluted at the end of the ammonium sulfate gradient (0.3-0M). Appropriate fractions were pooled and dialyzed against a buffer containing 50 mM NaOAc (pH=6), then passed through a porous 50HQ chromatography column. The HQ flow-through was diluted 4-fold and loaded onto a Toyopearl DEAD 650M chromatography column, then eluted with 25mM sodium citrate, 125M NaCl.

In another embodiment, recombinant Neutrokine-α was expressed and purified using a baculovirus vector system in Sf+ insect cells.

First, the Ser-78 to Leu-285 amino acid residues of the Neutrokine-α polypeptide sequence shown in Figure 1A and 1B (equal to the Ser-78 to Leu-285 amino acid residues of the Neutrokine-α polypeptide sequence shown in SEQ ID NO: 2 base), subcloned into the baculovirus transfer construct PSC to generate the baculovirus expression plasmid. The pA2GP transfer vector derived from pVL941, containing the gp67 signal peptide, a modified multiple cloning site, and the lac gene cloned downstream of the Drosophila heat shock promoter for selection of blue-stained plaques. Using the Neutrokine-α (SEQ ID NO: 2) sequence, and the pA2GP vector sequence, a cloning method was designed to tightly fuse the PSC signal peptide coding sequence to the Neutrokine-α coding sequence (SEQ ID NO: 2 and Figures 1A and 1B) Ala-134 and insert it into the PSC bacmid transfer plasmid. This method involves the use of two polymerase chain reactions (PCR). First, primers for amplifying the Neutrokine-α sequence were designed. The 5' primer consisted of the sequence encoding Ala-134 and the following residues (5'-GGT CGC CGT TTC TAA CGC GGCCGT TCA GGG TCC AGA AG-3'; SEQ ID NO: 31) at the C-terminus encoding the PSC signal peptide before the sequence. 3' primer (5'-CTG GTTCGG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTAGAT C-3'), consisting of the reverse complement of the pA2GP vector sequence immediately downstream of the Neutrokine-α coding sequence, at Kpn I It is preceded by a restriction endonuclease site and a spacer sequence (to improve KpnI cleavage efficiency). PCR was performed using pA2GP containing the Neutrokine-alpha plasmid template and primers O-1887 and O-1888, and the resulting PCR products were purified by standard methods.

Another PCR reaction was performed using the PSC baculovirus transfer plasmid pMGS12 as template. The pMGS12 plasmid consists of the AcNPV EcoRI "I" fragment inserted into pUC8, with the polyhedrin coding sequence after the ATG start codon replaced with the PSC signal peptide and a polylinker site. The PCR reaction used pMGS12 as a template, the 5' primer (5'-CTGGTA GTT CTT CGG AGT GTG-3'; SEQ ID NO: 33) annealed in AcNPV ORF603 upstream of the unique NgoMIV and EcoRV sites, and the 3' primer (5 '-CGCGTT AGA AAC GGC GAC C-3'; SEQ ID NO: 34) anneals to the 3' end of the sequence encoding the PSC signal peptide.

To generate a PCR product in which the PSC signal peptide was tightly fused to the Neutrokine-alpha coding sequence Ala-134, this PCR product was combined with the PSC signal peptide-polyhedrin upstream region PCR product and one more cycle of PCR was performed. Since the 3' end of the PSC signal peptide PCR product (pMGS12/O-959/O-1044) overlaps with the 5' end of the Neutrokine-α PCR product prepared with primers O-1887/O-1888, combine the two PCR products and use Primers O-959 and O-1888 were overlap extended by PCR.

The resulting overlap-extended PCR product, containing the PSC signal peptide fused to the Neutrokine-alpha sequence, was inserted into the baculovirus transfer plasmid pMGS12. This PCR product was digested with NgoM IV and Kpn I, and the fragment was purified and ligated into NgoM IV-Kpn I cut pMGS12. After transformation of competent E. coli DH5α cells with this ligation mixture, colonies were picked and plasmid DNA was minipreps. Some positive clones for each ligation reaction were identified by restriction digest analysis of plasmid DNA, and 3 clones (pAcC9669, pAcC9671, pAcC9672) were selected for large-scale plasmid purification. DNA sequence analysis was performed on the resulting plasmid DNA to identify and sequence the Neutrokine-alpha insert.

The following steps illustrate the recovery and purification of recombinant Neutrokine-α from Sf+ insect cells. Unless otherwise specified, the methods were carried out at 2-8°C.

Recycle

Step 1: CaCl ₂ treatment

Sf+ cell supernatants were collected by centrifugation at 8000 xg. Recovery Buffer-1 (1M CaCl ₂ ) was added to the supernatant so that the final concentration of CaCl ₂ was 10 mM. (In a preferred embodiment, 1M _ZnCl2 is used instead of 1M _CaCl2 ). The pH of this solution was adjusted to 7.7± with Recovery Buffer-2 (1M Tris pH8(±0.2)). The solution was incubated for 15 minutes and then centrifuged at 8000 xg.

purification

Step 1: Chromatography on a Porous PI-50 Column

The Sf+ cell supernatant was loaded on a porous PI-50 chromatography column (PE Biosystem), which was equilibrated with PI-1 buffer (50mM Tris, 50mM NaCl, pH7.4 (±0.2)). The PI-50 chromatography column was washed with 1-2CV of PI-1 buffer, and then eluted with PI-2 buffer (50 mM sodium citrate, pH 6 (±0.2)) with a linear gradient of 3 CV. Ultraviolet (UV) absorbance at 280 nm of the eluate was monitored. Fractions past the elution peak were collected and analyzed by SDS-PAGE. Assemble the appropriate components.

Step 2: Chromatography on Toyopearl Hexyl 650C column

The pooled PI was mixed with salt to a final concentration of (NH ₄ ) ₂ SO ₄ of 0.7 M, and loaded on a Toyopearl Hexyl 650C chromatography column with HIC-1 buffer (50 mM NaOAc, 0.6 M NaCl , 0.7M (NH ₄ ) ₂ SO ₄ pH 6 (±0.2)) equilibrium. Then the column was washed with 2CV of HIC-1 buffer. Next, recombinant Neutrokine-α was eluted with 3-5CV of HIC-2 buffer (50 mM NaOAc pH6 (±0.2)), and then washed with 2CV of 20% ethanol. Ultraviolet (UV) absorbance and conductivity at 280 nm of the eluate were monitored. Fractions of eluted peaks were collected and analyzed by SDS-PAGE. Assemble the appropriate components.

Step 3: Chromatography on SP Sepharose FF

The Hexyl fraction was dialyzed and adjusted to pH 4.5 with SP-1 buffer (50 mM sodium acetate, pH 4.5 (±0.2)), diluted 4-fold, and loaded on the SP Sepharose (cation exchanger, Pharmacia) layer The chromatography column was equilibrated with SP-1 buffer (50 mM sodium acetate, pH 4.5 (±0.2)). The recombinant Neutrokine-α protein was then eluted from the SP chromatography column at pH 5.5 with SP-2 buffer (50 mM sodium acetate, pH 5.5 (±0.2)). The eluate was then monitored for ultraviolet (UV) absorbance at 280 nm. Fractions past the elution peak were collected and analyzed by SDS-PAGE. Assemble the appropriate components.

Step 4: Dialysis of recombinant Neutrokine-α

The SP fraction was placed in a 6-8 kd cut-off membrane device, then dialyzed or diafiltered overnight against dialysis buffer (10 mM sodium citrate, 140 mM NaCl, pH 6 (±0.2)).

Step 5: Filter and Fill

The protein concentration of the recombinant Neutrokine-α solution in the sixth step was determined by bicinchoninic acid (BCA) protein analysis. Recombinant Neutrokine-α was adjusted to the final protein concentration with appropriate buffer and filtered under controlled conditions. Store the filtrate in a suitable sterile container below -20°C.

In a specific embodiment, the Neutrokine-alpha protein of the present invention produced as described above is adjusted to a final protein concentration of 1-5 mg/ml, and treated with 10 mM sodium citrate, 140 mM NaCl, pH=6.0 (±0.4) Buffer and store in Type 1 glass bottles below -20°C.

During chromatography, ultraviolet (UV) absorbance at 280 nm was monitored. Chromatographic intermediates were tested for conductivity, pH and monitored by SDS and/or RP-HPLC when applicable.

Clean the chromatography column and purification equipment and wash with 0.2 or 0.5M NaOH and deionized water, followed by 0.1 or 0.5M acetic acid. Rinse columns and purification equipment with deionized water and, if necessary, store in appropriate storage solutions. Prior to use, the device is equilibrated with an appropriate buffer, such as described herein or well known in the art.

In a preferred embodiment, 1M _ZnCl2 is used instead of 1M _CaCl2 in the first step of the recovery described above. Also, in this embodiment, a combination of _ZnCl2 and _CaCl2 may be used. Some combinations of 0.1M ZnCl ₂ and 0.9M CaCl ₂ can be used in the recovery method of recombinant Neutrokine-α protein, such as but not limited to 0.1M ZnCl ₂ and 0.9M CaCl ₂ , 0.2M ZnCl ₂ and 0.8M CaCl ₂ , 0.3M ZnCl ₂ and 0.7M CaCl ₂ , 0.4M ZnCl ₂ and 0.6M CaCl ₂ , 0.5M ZnCl ₂ and 0.5M CaCl ₂ , 0.6M ZnCl ₂ and 0.4M CaCl ₂ , 0.7M ZnCl ₂ and 0.3M CaCl ₂ , 0.8M ZnCl ₂ and 0.2M CaCl ₂ , 0.9M ZnCl ₂ and 0.1M CaCl ₂ etc. However, the presence of EDTA will inhibit the recovery process. Additionally, the presence of _ZnCl2 and/or _CaCl2 in Recovery Buffer 1 will induce the formation of large amounts of high molecular weight Neutrokine-α multimers.

Example 3: Cloning and expression of Neutrokine-α in mammalian cells

A typical mammalian expression vector contains a promoter element that mediates transcription initiation of the mRNA, a protein coding sequence, and signals required for transcription termination and transcript polyadenylation. Other elements include enhancers, Kozak sequences and intervening sequences flanked by RNA splice donor and acceptor sites. Efficient transcription can be achieved with the early and late promoters of SV40, the long terminal repeats (LTRs) of retroviruses such as RSV, HTLVI, HIVI and the early promoters of cytomegalovirus (CMV). However, cytokines (such as the human actin promoter) can also be used. Suitable expression vectors for use in the present invention include, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that can be used include human HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, dreaded QC1-3 cells, mouse L cells, Chinese hamster ovary (CHO) cells, and HEK 293 cells.

Alternatively, the gene may be expressed in an appropriate cell line containing the gene integrated into the chromosome. Co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows identification and isolation of transfected cells.

Transfected genes can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is used to reveal cell lines carrying hundreds or even thousands of copies of the corresponding gene. Another useful selectable marker is glutamine synthase (GS) (Murphy et al., J. Biol. Chem. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, cells are grown in selected media and the most resistant cells are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are commonly used for protein production.

Expression vectors pC1 and pC4 contain the strong promoter (LTR) of Rous sarcoma virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41 : 521-530 (1985)). Multiple cloning sites such as BamHI, XbaI and Asp718 with restriction enzyme cutting sites facilitate the cloning of the corresponding genes. The vector additionally contains the 3' intron of the murine preproinsulin gene, polyadenylation and termination signals.

Example 3(a): Cloning and expression in COS cells

The expression plasmid pNeutrokine-α-HA was generated by cloning a part of the deposited cDNA encoding the extracellular domain of the protein into the expression vector pcDNAI/Amp or pcDNAIII (available from Invitrogen). To produce a soluble secreted form of the polypeptide, the extracellular domain was fused to the secretory leader sequence of the human IL-6 gene.

The expression vector pcDNAI/Amp contains: (1) Escherichia coli replication origin that efficiently propagates in Escherichia coli and other prokaryotic cells; (2) ampicillin resistance gene for selection of prokaryotic cells containing plasmid; (3) Proliferating SV40 origin of replication; (4) CMV promoter, polylinker, SV40 intron; (5) some codons encoding the hemagglutinin fragment (i.e. HA tag to facilitate purification), followed by stop codon and polyadenylation The polyadenylation signal whereby the cDNA can be conveniently placed under the expression control of the CMV promoter and operably linked to the SV40 intron and polyadenylation signal via restriction sites within the polylinker. This HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as described by Wilson et al., Cell 37:767 (1984). Fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with antibodies recognizing the HA epitope. In addition, pcDNAIII contains a selectable neomycin marker.

The DNA fragment encoding the extracellular domain of the Neutrokine-α polypeptide was cloned into the polylinker region of the vector, so that the expression of the recombinant protein was directed by the CMV promoter. The plasmid construction strategy is as follows. The Neutrokine-α cDNA of the deposited clone was amplified with primers containing conventional restriction sites, mostly as described above in the method for constructing a vector expressing Neutrokine-α in Escherichia coli. Suitable primers include those used in this example below. Contains the underlined BamHI site, the Kozak sequence, the AUG start codon, the sequence encoding the secretory leader peptide of the human IL-6 gene, and the 18 nucleotides 5' of the 5' coding sequence of the ectodomain of the Neutrokine-α protein Primer with the following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTCGGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTGCCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GACCTG GCC AGC- 3' (SEQ ID NO: 16). Contains an underlined BamHI restriction site, and an 18 nucleotide 3' primer complementary to the 3' coding sequence preceding the stop codon, with the following sequence: 5'-GTG GGA TCC TTA CAG CAG TTTCAA TGC ACC-3 ' (SEQ ID NO: 17).

The DNA fragment amplified by PCR and the vector pcDNAI/Amp were digested with BamHI and ligated. This ligation mixture was transformed into E. coli strain SURE (purchased from Stratagene Cloning Systems, 11099North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture was plated on ampicillin medium, and then the plate was incubated to make ampicillin Growth of penicillin-resistant colonies. Isolate plasmid DNA from resistant colonies, and detect the presence of fragments encoding Neutrokine-α ectodomain by restriction analysis or other methods.

For expression of recombinant Neutrokine-α, COS cells are transfected with the expression vector as described above, using DEAE-DEXTRAN, such as described by Sambrook et al., Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) . Cells were incubated under conditions expressing Neutrokine-α by the vector.

Expression of the Neutrokine-[alpha]-HA fusion protein was detected by radiolabeling and immunoprecipitation as described by Harlow et al., Antibody Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). Two days after transfection, cells were labeled by incubation in medium containing ³⁵ S cysteine for 8 hours. Collect cells and medium, wash and lyse cells with RIPA buffer containing detergent: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as As described by Wilson et al. above. Proteins were precipitated from cell lysates and media with HA-specific monoclonal antibodies. Precipitated proteins were then analyzed by SDS-PAGE and autoradiography. Expression products of expected size were seen in cell lysates but not in negative controls.

Example 3(b): Cloning and expression in CHO cells

Vector pC4 was used to express Neutrokine-α protein. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC Accession No: 37146). To produce a soluble secreted form of the polypeptide, the extracellular domain was fused to the secretory leader sequence of the human IL-6 gene. The vector plasmid contains the murine DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary or other cells lacking dihydrofolate activity transfected with these plasmids can be selected by growing the cells in selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate . Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (see e.g. Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biochem. 253: 1357-1370, Hamlin, J.L. and Ma, C. 1990, Acta Biochem. Biophysiol. 1097:107-143, Page, M.J. and Sydenham, M.A. 1991, Biotechnology 9:64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the targeted enzyme DHFR with consequent amplification of the DHFR gene. If another gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this method can be used to generate cell lines carrying more than 1000 copies of the amplified gene. Next, when the methotrexate is removed, a cell line is obtained that contains the amplified gene integrated into one or more chromosomes of the host cell.

Plasmid pC4 contains a strong promoter for the long terminal repeat (LTR) of Rouse sarcoma virus expressing the corresponding gene (Cullen et al., Mol. CMV) fragment of the enhancer of the immediate early gene (Boshart et al., Cell 41:521-530 (1985)). Downstream of the promoter is a restriction enzyme cleavage site that allows integration of the gene: BamHI, XbaI, and Asp718. Following these cloning sites, the plasmid contains the 3' intron and polyadenylation site of the murine preproinsulin gene. Other high-efficiency promoters can also be used for expression, such as the human β-actin promoter, the SV40 early or late promoter, or the long terminal repeats of other retroviruses such as HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express Neutrokine-α in a regulated manner in mammalian cells (Gossen, M., & Bujard, H.1992, PNAS 89:5547- 5551). Other signals for polyadenylation of mRNA, for example the human growth hormone or globin genes can be used. Stable cell lines carrying the corresponding genes integrated into the chromosome can also be selected on the basis of co-transfection with selectable markers such as gpt, G418 or hygromycin. It is advantageous to start with more than one marker such as G418 plus methotrexate.

Plasmid pC4 was digested with the restriction enzyme BamHI and then dephosphorylated with calf intestinal phosphatase by methods known in the art. The vector was then isolated from a 1% agarose gel.

The DNA sequence encoding the ectodomain of the Neutrokine-alpha protein was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. Contains the underlined BamHI site, the Kozak sequence, the AUG start codon, the sequence encoding the secretory leader peptide of the human IL-6 gene, and the 18 nucleotides 5' of the 5' coding sequence of the ectodomain of the Neutrokine-α protein Primer with the following sequence: 5'-GCG GGA TCC GCC ACC ATGAAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTCTCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCTGCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC- 3' (SEQ ID NO: 16). Contains an underlined BamHI restriction site, and an 18 nucleotide 3' primer complementary to the 3' coding sequence preceding the stop codon, with the following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC- 3' SEQ ID NO: 17).

The amplified fragment was digested with endonuclease BamHI and repurified on a 1% agarose gel. This isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed, and bacteria containing the fragment inserted into plasmid pC4 are identified, for example, by restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene were used for transfection. 5 μg of expression plasmid pC4 was co-transfected with 0.5 μg of plasmid pSVneo by lipofection (Felgner et al., supra). Plasmid pSV2-neo contains a dominant selectable marker encoding the neo gene for antibiotic resistance-conferring enzymes including Tn5 of G418. Cells were seeded in α-minus MEM medium supplemented with 1 mg/ml G418. After 2 days, cells were trypsinized and seeded on hybridoma cloning plates (Greiner, Germany) in α-minus MEM medium supplemented with 10, 25, or 50 ng/ml methotrexate and 1 mg/ml G418. After about 10-14 days, individual clones were digested with trypsin, and seeded in 6-well culture dishes or 10 ml culture flasks with different concentrations of methotrexate (50, 100, 200, 400, 800 Nm). Clones grown in the highest concentration of methotrexate were then transferred to new 6-well plates containing higher concentrations of methotrexate (1, 2, 5, 10, 20 [mu]M). The same steps were repeated until a clone growing at a concentration of 100-200 μM was obtained. Expression of the desired gene product is analyzed by, for example, SDS-PAGE and Western blot or by reverse phase HPLC.

The inventors have generated at least six Neutrokine-alpha expression constructs to facilitate production of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides in different formats and in some systems. This expression construct is as follows: (1) pNa.A71-L285 (express Ala71-Leu285 amino acid residues), (2) pNa.A81-L285 (express Ala81-Leu285 amino acid residues), (3) pNa. L112-L285 (expressing amino acid residues at position Leu112-Leu285), (4) pNa.A134-L285 (expressing amino acid residues at position Ala134-Leu285), (5) pNa.L147-L285 (expressing amino acid residues at position Leu147-Leu285 ), (6) pNa.G161-L285 (expressing Gly161-Leu285 amino acid residues).

In preferred embodiments, expression constructs are used to express various Neutrokine-alpha muteins from bacterial, baculoviral and mammalian systems.

In other preferred embodiments, the construct expresses a Neutrokine-α polypeptide fragment fused to a heterologous polypeptide at the N or C terminus, such as the signal peptide of human IL-6, the signal peptide of CK-β8 ( -21 to -1 amino acids of the CK-β8 sequence disclosed in PCT publication PCT/US95/09058), or the human IgG Fc region. Other sequences that can be used are known to those skilled in the art.

Example 4: Tissue distribution of Neutrokine-α mRNA expression

Northern blot analysis was performed to examine the expression of the Neutrokine-α gene in human tissues using the method as described by Sambrook et al. A cDNA probe containing the entire nucleotide sequence of Neutrokine-α protein (SEQ ID NO: 1) was labeled with ³² p using the rediprime ^™ DNA labeling system (Amersham Life Science) according to the manufacturer's instructions. After labeling, the probe was purified using a CHROMA SPIN-100 ^™ column (Clontech Laboratories, Inc.) according to the manufacturer's protocol number PT1200-1. The purified labeled probes were then used to detect Neutrokine-α and/or Neutrokine-αSV mRNA in various human tissues.

Multitissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech using ExpressHyb ^™ Hybridization Solution (Clontech) labeled with Probe detection. After hybridization and washing, blots were exposed to film overnight at -70°C, which was developed according to standard methods.

To determine the expression pattern of Neutrokine-[alpha] and/or Neutrokine-[alpha]SV, a panel of multi-tissue Northern blots was probed. It showed that a 2.6kb mRNA was significantly expressed in peripheral blood leukocytes, spleen, lymph nodes and bone marrow, and could be detected in placenta, heart, lung, fetal liver, thymus and pancreas. Analysis of a panel of cell lines showed that Neutrokine-α and/or Neutrokine-αSV were highly expressed in HL60 cells and detectable in K562 cells, but not in Raji, HeLa, or MOLT-4 cells. All analyzes showed enrichment of Neutrokine-α and/or Neutrokine-αSV mRNA expression in the immune system.

Example 5: Gene therapy with endogenous Neutrokine-α gene

According to another kind of gene therapy of the present invention, comprise endogenous Neutrokine-alpha sequence and promotor operably combined by homologous recombination, as US Patent No: 5641670 authorized on June 24, 1997; International Publication WO 96 /29411, published September 26, 1996; International Publication WO 94/12650, published August 4, 1994; Koller et al., Proceedings of the National Academy of Sciences 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435 -438 (1989). This method involves activating a gene that is present in the target cell, but is not expressed or expressed at a low level in the cell. A polynucleotide construct containing a promoter and targeting sequences homologous to the 5' noncoding sequences of endogenous Neutrokine-[alpha] flanking the promoter was generated. The targeting sequence is near the 5' end of Neutrokine-α, whereby the promoter is operably linked to the endogenous sequence by homologous recombination. Promoter and targeting sequences can be amplified by PCR. Preferably, the amplified promoter contains unique restriction enzyme sites at the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction enzyme site as the 3' end of the amplified promoter. the same restriction enzyme sites.

The amplified promoter and amplified targeting sequence were digested with appropriate restriction enzymes and then treated with calf intestinal phosphatase. The digested promoter was added together with the digested targeting sequence in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The constructs were size fractionated on agarose gels and purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide constructs are administered as naked polynucleotides by electroporation. However, polynucleotide constructs can also be administered with transfection-promoting agents, such as liposomes, viral sequences, viral particles, precipitants, and the like. Such delivery is known in the art.

Once the cells are transfected, homologous recombination is performed so that the promoter is operably linked to the endogenous Neutrokine-alpha sequence. This enables the expression of Neutrokine-α in the cells. Expression can be detected by immunostaining or any other method known in the art.

Fibroblasts are obtained from skin biopsies of subjects. The resulting tissue was placed in DMEM+10% fetal bovine serum. Exponentially growing or early stationary-phase fibroblasts were trypsinized and rinsed from the plastic surface with nutrient medium. A portion of the cell suspension was removed for counting and the remaining cells were centrifuged. The supernatant was aspirated and the pellet was resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 _{mM Na2HPO4} , 6 mM glucose). The cells were re-centrifuged, the supernatant was aspirated, and the cells were resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contained approximately 3 x ¹⁰⁶ cells/ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA was prepared according to standard methods. For example, to construct a plasmid directed to the Neutrokine-alpha locus, plasmid pUC18 (MBI Fermentans, Amherst, NY) was digested with HindIII. The CMV promoter was amplified by PCR to contain an XbaI site at the 5' end and a BamHI site at the 3' end. Two Neutrokine-α non-coding sequences were amplified by PCR: one Neutrokine-α non-coding sequence (Neutrokine-α fragment 1) was amplified to contain a HindIII site at the 5' end and an XbaI site at the 3' end; the other The Neutrokine-α non-coding sequence (Neutrokine-α fragment 2) contains a BamHI site at the 5' end and a HindIII site at the 3' end after amplification. CMV promoter and Neutrokine-α fragment were digested with appropriate enzymes (CMV promoter was digested with XbaI and BamHI; Neutrokine-α fragment 1 was digested with XbaI; Neutrokine-α fragment 2 was digested with BamHI) and ligated together. The resulting ligation product was digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA was added to sterile test tubes (Bio-Rad) with a 0.4 cm electrode gap. The final DNA concentration was at least 120 μg/ml. Then 0.5 ml of cell suspension (containing approximately 1.5 x 10 ⁶ cells) was added to the test tube, and the cell suspension and DNA solution were mixed slightly. Electroporation was performed with a Gene-Pulser instrument (Bio-Rad). Capacitance and voltage were set to 960μF and 250-300V, respectively. As the voltage was increased, cell survival decreased, but the percentage of surviving cells stably incorporating the introduced DNA into their genome increased significantly. Given these parameters, pulse times of approximately 14-20 milliseconds should be observed.

The electroporated cells were kept at room temperature for approximately 5 minutes before the contents of the tubes were gently removed with a sterile pipette. Cells were added directly to 10 ml of pre-warmed nutrient medium (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37°C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and incubated for an additional 16-24 hours.

The genetically engineered fibroblasts are then injected into the host, either alone or after growing to confluence with cytodex 3 microcarrier beads. Fibroblasts now produce protein products. The fibroblasts can then be introduced into the patient, as described above.

Example 6: Neutrokine-α, a new member of the tumor necrosis factor ligand family as a B lymphocyte stimulator

In a human neutrophil/monocyte-derived cDNA library, a 285 amino acid protein was identified that, in its putative extracellular receptor-ligand binding domain, was associated with APRIL (28.7%) ( Hahne, M. et al., J. Experimental Methods 188, 1185-90 (1998)), TNF-α (16.2%) (Pennica, D. et al., Nature 312, 724-729 (1984)) and LT-α (14.1%) ) (Gray, Nature 312, 721-724 (1984)) exhibited a clear homology (Fig. 7A). We call this cytokine Neutrokine-alpha (based on its biological activity, it is also called B lymphocyte stimulator (BlyS)). Hydrophobicity analysis of the Neutrokine-α protein sequence revealed that a potential transmembrane domain between amino acid residues 47-73 preceded non-hydrophobic amino acids, suggesting that, like other TNF ligand families, Neutrokine-α is a A type II annexin (Cosman, D., Stem Cell 12:440-55 (1994)). Expression of this cDNA in mammalian cells (HEK293 and Chinese hamster ovary cells) and Sf9 insect cells identified a soluble form of 152 amino acids with an N-terminal sequence starting at alanine 134 (Fig. 7A). Reconstruction of the mass and charge ratios determined the molecular weight of Neutrokine-α to be 17038 Daltons, a value consistent with the putative molecular weight of this 152 amino acid protein with one disulfide bond (17037.5 Daltons).

Using mapping of human/hamster somatic hybrids and radiation hybrids, the gene encoding Neutrokine-α was found to be linked to marker SHGC-36171, which localized to human chromosome 13q34, a region unrelated to any other member of the TNF superfamily of genes ( Cosman, D. Stem Cells 12:440-55 (1994)).

The expression pattern of Neutrokine-α was determined by Northern blot (Fig. 7B) and flow cytometric analysis (Table 5 and Fig. 8). Neutrokine-α is encoded by a 2.6kb mRNA, which is found to be highly expressed in peripheral blood leukocytes, spleen, lymph nodes and bone marrow. Low level expression was detected in placenta, heart, lung, fetal liver, thymus and pancreas. In this panel of cell lines, Neutrokine-α mRNA was detected in HL-60 and K562, but not in Raji, HeLa, or MOLT-4 cells. These results were corroborated by flow cytometric analysis using Neutrokine-α specific mAb 2E5. As shown in Figure 5, Neutrokine-α expression was not detected on T or B cell lines, but was restricted to cells of myeloid origin. Further analysis of normal blood cells showed significant expression on quiescent monocytes and an approximately 4-fold upregulation after exposing cells to IFN-γ (100 U/ml) for 3 days (Fig. 8A). A concomitant increase in Neutrokine-α-specific mRNA was also detected (Fig. 8B). In contrast, Neutrokine-α was not expressed in freshly isolated peripheral blood granulocytes, T cells, B cells or NK cells.

To determine the ability of purified recombinant Neutrokine-α (rNeutrokine-α) to induce activation, proliferation, differentiation or death of cells including B cells, T cells, monocytes, NK cells, Hematopoietic progenitor cells, and cells of various endothelial and epithelial origins. In these assays, Neutrokine-α was specifically found to increase B cell proliferation in standard co-stimulation assays in which purified tonsillar B cells, in the presence of formalin-fixed S. aureus Cowan I (SAC) or fixed anti-human Incubate with IgM as the elicitor (Sieckmann, D.G., et al., J. Experimental Methods 147:814-29 (1978); Ringden, O. et al., Scand. J. Immunol. 6:1159-69 (1977)). As shown in Figure 9A, recombinant Neutrokine-α induced dose-dependent proliferation of tonsillar B cells. This response was similar to that of rIL2 in the dose range of 0.1-10000 ng/ml. Neutrokine-α also induced B cell proliferation when cell cultures were co-stimulated with immobilized anti-IgM antibodies (Fig. 9B). In the presence of fixed concentrations of IL2 or rNeutrokine-[alpha], a dose-dependent response was readily observed as the amount of cross-linker was increased.

In an attempt to link specific biological activity on B cells to receptor expression, purified Neutrokine-α was biotinylated. The resulting biotinylated Neutrokine-α retained biological function in standard B cell proliferation assays. Lineage-specific analysis of human peripheral whole blood cells showed that binding of biotinylated Neutrokine-α was undetectable on T cells, monocytes, NK cells and granulocytes, as determined by CD3, CD14, CD56 and CD66b, respectively (FIG. 10A). In contrast, biotinylated Neutrokine-α bound peripheral blood CD20+ B cells. Receptor expression was also detected in B-cell tumor lines REH, ARH-77, Raji, Namalwa, RPMI8226, and IM-9, but not in any of the bone marrow-derived cell lines tested including THP-1, HL-60, K-562 and U-937 were not detected. Representative flow cytometry profiles for the myeloma cell line IM-9 and the tissue cell line U-937 are shown in Figure 10B. Similar results were obtained by substituting biologically active FLAG-tagged Neutrokine-α protein for chemically modified biotinylated Neutrokine-α. These results confirm that Neutrokine-α exhibits distinct B-cell tropism both in its receptor distribution and biological activity. These results also show whether cellular activation can induce Neutrokine-alpha receptor expression in peripheral blood cells, other normal cells or established cell lines.

To test the species specificity of Neutrokine-α, mouse splenic B cells were cultured in the presence of human Neutrokine-α and SAC. The results showed that rNeutrokine-α induced the proliferation of murine splenic B cells in vitro and bound to cell surface receptors on these cells. Interestingly, immature surface Ig-negative B cell precursors isolated from mouse bone marrow neither proliferated in response to Neutrokine-α nor bound ligand.

To determine the in vivo activity of rNeutrokine-α, BALB/c mice (3/group) were injected (i.p.) twice a day with buffer, or 0.08mg/kg, 0.8mg/kg, 2mg/kg or 8mg/kg rNeutrokine-alpha. Mice were subjected to this treatment for 4 consecutive days, after which they were sacrificed and various tissues and sera were collected for analysis. In another embodiment, BALB/c mice are injected (i.p.) twice daily with rNeutrokine-α at any dose in the range of 0.01-10 mg/kg. In a preferred embodiment, BALB/c mice are injected (i.p.) twice a day with any dose of rNeutrokine-α in the range of 0.01-3 mg/kg (particularly preferred doses in this embodiment include but are not limited to 0.01 mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2 mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2.1mg/kg, 2.2 mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, and 3.0mg/kg). In another preferred embodiment, BALB/c mice are injected (i.p.) twice a day with any dose of rNeutrokine-α in the range of 0.02-2 mg/kg (particularly preferred doses in this embodiment include, but are not limited to 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg).

The effect of Neutrokine-[alpha] administration was evident in microscopically HE-stained spleen tissue sections and immunohistochemically stained with mAb specific for CD45R(B220) ( FIG. 11A ). Normal splenic architecture was altered, with marked extension of the white matter marginal zone and marked increase in red pulp cellularity (Fig. 11A). The marginal zone extension was due to increased numbers of lymphocytes expressing the B-cell marker CD45R (B220). In addition, the T cell-dense periarterial lymphatic sheath (PALS) area was also infiltrated by a moderate amount of CD45R(B220) positive cells. This suggested that white matter changes were due to increased numbers of B cells. The densely packed cell population that normally fills the red pulp is not stained by CD45R (B220). Additional experiments are required to characterize all cell types involved and to further determine the mechanism by which 0.08 mg/kg, 0.8 mg/kg, and 2 mg/kg alter spleen structure.

Flow cytometric analysis of spleen tissues from mice treated with 2 mg/kg Neutrokine-α showed that Neutrokine-α increased the proportion of mature (CD45R(B220) ^dull , ThB ^bright ) B cells by nearly 10x (Fig. 11B). In addition, the analysis in which mice were treated with buffer, 0.08mg/kg, 0.8mg/kg, 2mg/kg, or 8mg/kg of Neutrokine-α showed that compared with control mice, 0.08mg/kg, 0.8 mg/kg, 2mg/kg Neutrokine-α both increased the proportion of mature (CD45R(B220) ^dull , ThB ^bright ) B cells nearly 10 times, while buffer and 8mg/kg Neutrokine-α produced about the same proportion of mature B cells . See Table 4.

Table 4: FACS analysis of mouse splenic B cell populations

Neutrokine-α (mg/kg) Percentage of mature B cells (R2) Percentage of positive CD45R (R1)

Control (buffer) 1.26 52.17

0.08mg/kg 16.15 56.53

0.8mg/kg 18.54 57.56

2mg/kg 16.54 57.55

8mg/kg 1.24 61.42

A potential consequence of increased expressivity of mature B cells in vivo is a relative increase in serum Ig titers. Therefore, serum IgA, IgG and IgM levels were compared between buffer and Neutrokine-α treated mice (Fig. 11C). Administration of Neutrokine-α increases serum IgA and IgM levels by 2 and 5 fold, respectively. Remarkably, circulating levels of IgG did not increase.

In addition, in the serum IgA titration of mice treated with various amounts of Neutrokine-α for 4 days, a dose-dependent response was observed, whereas no significant dose-dependence was observed for 2 days of administration of the same amount of Neutrokine-α. In the case of administration for 4 days, the serum IgA titers caused by administration of 8, 2, 0.8, 0.08 and 0 mg/kg Neutrokine-α were approximately 800 μg/ml, 700 μg/ml, 400 μg/ml, 200 μg/ml, and 200 μg/ml ml. That is, after 4 days of administration of 8, 2, 0.8, and 0.08 mg/kg of Neutrokine-α, compared with the background or basal IgA serum levels observed with buffer alone, the IgA serum levels were increased by approximately 4, 3.75, 2, and minimal folds, respectively . In an additional embodiment, these assays can be performed with any amount of rNeutrokine-[alpha] in the range of 0.01-10 mg/kg. In a preferred embodiment, Neutrokine-α in the range of 0.01-3 mg/kg is administered (in this embodiment, particularly preferred doses include but are not limited to 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg , 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg , 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg , 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg , 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, 3.0mg/kg). In another preferred embodiment, Neutrokine-α in the range of 0.02-2 mg/kg is administered (in this embodiment, particularly preferred doses include but are not limited to 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg).

The evidence presented here clarifies that Neutrokine-α is a new member of the TNF-ligand superfamily that induces B cell proliferation and differentiation both in vivo and in vitro. Neutrokine-α differs from other B cell growth and differentiation factors such as IL2 by its monocyte-specific gene/protein expression pattern, and its specific receptor distribution and biological activity on B lymphocytes (Metzger, DW et al., Immuno Scientific Research 146: 499-505 (1995)), IL4 (Armitage, RJ et al., Advances in Biological Experimental Methods 292: 121-30 (1991); Yokota, T. et al., Proceedings of the American Academy of Sciences 83: 5894-98 (1986 )); IL5 (Takatsu, K. et al., Proceedings of the National Academy of Sciences 84: 4234-38 (1987); Bertolini, JN et al., European Journal of Immunology 23: 398-402 (1993)), IL6 (Poupart, P. et al. , EMBO Journal 6: 1219-24 (1987); Hirano, T., Nature 324: 73-76 (1986)), IL7 (Goodwin, RG et al., Proceedings of the National Academy of Sciences 86: 302-06 (1989); Namen, AE et al., Nature 333:571-73 (1988)), IL13 (Punnonen, J. et al., Allergy 49:576-86 (1994)), IL15 (Armitage, RJ et al., J. Immunol. 154:483-90 ( 1995)), CD40L (Armitage, RJ et al., Nature 357:80-82 (1992); Van Kooten, C. and Banchereau, J.Int, Arch.Allergy. Immunol 113:393-99 (1997)) or CD27L ( CD70) (Oshima, H. et al., Int. Immunol. 10:517-26 (1998); Lens, SM et al., Semin. Immunol. 10:517-26 (1998)). These arguments suggest that Neutrokine-α is involved in the signal exchange between B cells and monocytes or their differentiated progeny. Although this signaling mode is available to all B cells, this restricted expression pattern and Ig secretion suggest a role for Neutrokine-α in the activation of CD5 ⁺ or "unconventional" B cell responses. These B cells provide an important component of the innate immune system and provide protection against pathogens in the environment through their secretion of polyreactive IgM and IgA antibodies (Pennell, CA et al., European Journal of Immunology 19: 1289-95 (1989) ; Hayakawa, K. et al., PNAS 81: 2494-98 (1984)). Alternatively, Neutrokine-α may act as a modulator of T-cell-independent responses in the same manner as CD40 and CD40L in T-cell-dependent antigen activation (Eertwegh, AJ et al., J. Experimental Methods 178:1555-65( 1993); Grabstein, KH et al., J. Immunol. 150:3141-47 (1993)). Thus, Neutrokine-alpha, its receptor or related antagonists are useful in the treatment of B cell disorders associated with autoimmunity, neoplasia and/or immunodeficiency syndromes.

method

Mice: BALB/cAnNCR (6-8 weeks) were purchased from Charles River Laboratories and housed in small isolation cages with reusable paper pads according to recommended standards (National Research Council, and Guidelines for the Use of Laboratory Animals (1999)). (Harlan Sprague Dawley, Indianapolis, IN) and were provided with pelleted rodent chow (Harlan Sprague Dawley), and bottled drinking water ad libitum at the bottom. The animal protocol used in this study was referenced and approved by the HGS Institutions of Animal Care and Use Committee.

Isolation of cDNA for full-length Neutrokine-α: The Human Genome Sciences Inc. Expressed Sequence Tag (EST) database was searched for sequences homologous to the receptor binding domain of the TNF family using the BLAST program. The full-length Neutrokine-α clone was identified, sequenced and submitted to GenBank (Accession No. AF132600). The Neutrokine-α open reading frame was amplified by PCR using 5'primers and 3'primers. The 3' primer (5'-CAG ACT GGT ACC GTCCTG CGT GCA CTA CAT GGC-3') anneals at the predetermined downstream stop codon. The resulting amplicons were tailed with BamHI and Asp718 restriction sites and subcloned into mammalian expression vectors. Neutrokine-α was also expressed in p-CMV-1 (Sigma Chemicals).

Purification of recombinant human Neutrokine-α: The full-length cDNA encoding Neutrokine-α was subcloned into baculovirus expression vector pA2 and transfected into Sf9 insect cells (Pater.V.P. et al., Journal of Expression Methods 185:1163-72 (1997)). Recombinant Neutrokine-α was purified from cell supernatant 92 hours post-infection using a combination of anion exchange, size exclusion and hydrophobic interaction chromatography. Purified protein was formulated in buffer containing 0.15M NaCl, 50mM NaOAc, pH 6, sterile filtered and stored at 4°C until required. Both SDS-PAGE and RP-HPLC analysis showed that rNeutrokine-α was more than 95% purified. Endotoxin levels were below the limit of detection in the LAL assay (Associates of Cape Cod, Falmouth, MA). The N-terminal sequence of the finally purified Neutrokine-α protein is Ala-Val-Gln-Gly-Pro. This corresponds to the sequence of soluble Neutrokine-α derived from a CHO cell line stably transfected with the full-length Neutrokine-α gene.

Generation of monoclonal antibodies: BALB/cAnNCR mice were immunized with 50 μg of His-tagged Neutrokine-α suspended in complete Freund's adjuvant, followed by two challenges in incomplete Freund's adjuvant. Hybridomas and monoclonal antibodies were prepared as described (Gefter, M.L. et al., Somatic. Cbll Genet. 3:231-36 (1977); Akerstrom, B. et al., J. Immunol. 135:2589-92 (1985)).

Cell lines: All human cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA).

FACS analysis: Neutrokine-α expression in human cell lines, freshly isolated normal peripheral blood nucleated cells, and monocytes cultured in vitro, mouse anti-human Neutrokine-α mAb 2E5 (IgG1), and mouse IgG PE conjugated F(ab')2 goat antibody (CALTAG Laboratories, Burlingame, CA) was evaluated. Cells were analyzed with FACScan (Becton Dickinson Immunocytometry System, San Jose, CA), and dead cells were excluded with propidium iodide. Neutrokine-α binding was determined with rNeutrokine-α biotinylated with N-hydroxysuccinimide biotin reagent (Pierce, Rockford, IL), and PE-conjugated streptavidin (Dako Corp, Glostrup, Denmark) .

Chromosome mapping: To determine the chromosomal location of the Neutrokine-α gene, a group of monochromosomal somatic cell hybrids (Quantum Biotechnology, Canada) that retain individual chromosomes were screened by PCR with Neutrokine-α-specific primers (5' primer: 5' -TGG TGT CTT TCT ACC AGG TGG-3'), 3' primer: 5'-TTTCTT CTG GAC CCT GAA CGG-3'). The putative 233bp PCR product was detected only in human chromosome 13 hybrids. A set of 83 radiation hybrids (ResearchGenetics, St. Louis, MO) and the Stanford Human Genome Center database ( http://www.shgc.stanford.edu.RH/rhserver ) were used. Neutrokine-α was found to be linked to the SHGC-36171 marker on chromosome 13. The cytogenesis map of human chromosome 13 was used to supermap this map to confirm that human Neutrokine-α was on chromosome band 13q34.

B Lymphocyte Proliferation Assay: Human tonsillar B cells were purified by depletion of CD3 positive cells with magnetic beads (MACS). The resulting cell population was more than 95% B cells as determined by the expression of CD19 and CD20. Place various dilutions of human rNeutrokine-α or control protein recombinant human IL2 in each well of a 96-well plate, add ¹⁰⁵ B cells suspended in culture medium, the total volume is 150 μl, and the culture medium used is RPMI1640 containing 10% FBS, 5×10 ⁻⁵ M 2ME, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 ⁻⁵ Pansorbin (SAC) or anti-IgM antibody. Proliferation was quantified by a 20 hour pulse (1 [mu]Ci/well) ^of3H -thymidine (6.7Ci/Mm) 72 hours after addition of the above factors.

Histological analysis: spleen tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 5 μm sections, placed on glass slides, and stained with HE or by enzymatic analysis of CD45R (B220) Indirect immunohistochemical staining of markers (Hilbert, D.M., Eur. J. Immunol. 23:2412-18 (1993)).

Table 5: Cell surface expression of Neutrokine-α

Example 7: Assays for Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation

The generation of a functional humoral immune response requires soluble and correlated signals between the B cell lineage and its microenvironment. The signal can give a positive stimulus, allowing cells of the B-cell lineage to continue their programmed development, or a negative stimulus, directing the cell to inhibit its current developmental pathway. A number of stimulatory and inhibitory signals have been found to affect B cell responses, including IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL14 and IL15. Interestingly, these signals are weak effectors by themselves but can combine with various co-stimulatory proteins to induce activation, proliferation, differentiation, homing, tolerance and death of B cell populations. One of the best B cell co-stimulatory proteins is the TNF superfamily. CD40, CD27 and CD30 in this family and their respective ligands CD154, CD70 and CD153 have been found to regulate various immune responses. Assays that detect and/or observe the proliferation and differentiation of these B cell populations and their precursors are very useful in determining the effects that various proteins may have on these B cell populations during proliferation and differentiation. Listed below are two assays that detect differentiation, proliferation or inhibition of B cell populations and their precursors.

In vitro assay: To determine the ability of purified Neutrokine-α and/or Neutrokine-αSV protein, or truncated forms thereof, to induce activation, proliferation, differentiation or inhibition and/or death of B cell populations and their precursors. The effect of Neutrokine-α and/or Neutrokine-αSV protein on purified human tonsil B cells, quantified over a dose range of 0.1-10,000 ng/ml, was determined in standard B lymphocyte co-stimulation in the presence of purified tonsil B cells Formalin-fixed Staphylococcus aureus Cowan I (SAC), or immobilized anti-human IgM antibody was cultured under the condition of triggering agent. Secondary signals such as IL-2 and IL-15 cooperate with SAC and IgM cross-linking to stimulate B cell proliferation measured by incorporation of tritiated thymidine. New synergists can be readily identified using this assay. This assay involved the isolation of human tonsillar B cells by magnetic bead (MACS) depletion of CD3 positive cells. The resulting cell population was more than 95% B cells as determined by the expression of CD45R(B220). Various dilutions of each sample were placed in each well of a 96-well plate, and ¹⁰⁵ B cells suspended in culture medium were added to each well, with a total volume of 150 μl. The culture medium used was 10% FBS, 5 × 10 ^-5 M of 2ME, 100 U/ml penicillin, 100 μg/ml streptomycin, and RPMI 1640 of 10 ⁻⁵ Pansorbin (SAC). Proliferation was quantified by a 20 hour pulse (1 [mu]Ci/well) ^of3H -thymidine (6.7Ci/Mm) 72 hours after addition of the above factors. Positive and negative controls were IL2 and medium, respectively.

Agonists (including Neutrokine-[alpha] and/or Neutrokine-[alpha]SV polypeptide fragments) demonstrate increased B cell proliferation when compared to what is observed when the same number of B cells are contacted with the same concentration of elicitor. Antagonists according to the invention exhibit reduced B-cell proliferation when compared to a control group containing the same number of B-cells, the same concentration of elicitor, and the same concentration of a soluble form of Neutrokine- α (such as amino acids 71-285, 81-285, 112-285 or 134-285 of the Neutrokine-α polypeptide shown in SEQ ID NO: 2), no antagonist.

In vivo analysis: BALB/c mice were injected (i.p.) twice daily with buffer, or 2 mg/kg of Neutrokine-α and/or Neutrokine-αSV protein, or a truncated form thereof. Mice received this treatment for 4 consecutive days before being sacrificed and various tissues and sera were collected for analysis. To compare normal HE tissue sections with Neutrokine-α and/or Neutrokine-αSV protein-treated spleen tissue, to identify the results of the effect of Neutrokine-α and/or Neutrokine-αSV protein on splenocytes, such as the spread of periarterial lymphatic sheaths, and And/or a marked increase in nucleated cellular structures in the red pulp area, which may indicate activation of differentiation and proliferation of B-cell populations. Immunohistochemical studies using the B cell marker, anti-CD45R (B220), to determine whether any physiological changes in splenocytes, such as changes in spleen histology, are due to infiltration within the loosely defined B cell zone of the established T cell zone Increased expression of B cells.

Flow cytometric analysis of spleen tissue from mice treated with Neutrokine-α and/or Neutrokine-αSV protein to indicate whether Neutrokine-α and/or Neutrokine-αSV specifically increased ThB+ , the proportion of CD45R(B220) null B cells.

In addition, a putative consequence of increased expression of mature B cells in vivo is a relative increase in serum Ig titers. Therefore, serum IgM and IgA levels were compared between buffer and Neutrokine-α and/or Neutrokine-αSV protein treated mice.

Example 8: Effects of Neutrokine-α and its agonists in the treatment of graft-versus-host disease-associated lymphoid atrophy and hypoplasia in mice

Analysis of Neutrokine-α Treatment, Prevention and/or Diagnosis of Graft Versus Host Disease (GVHD)-Associated Lymphoid Atrophy and Agenesis in Mice by Transplantation of C57BL/6 Parents into (BALB/c X C57BL/6)F1 (CBF1) mouse model. This parental entry into the F1 mouse model is a well-characterized, reproducible animal model of GVHD in bone marrow transplant patients, which is well known to those skilled in the art (see Gleichemann et al., Current Immunology 5:324, 1984). Soluble Neutrokine-α is expected to induce proliferation and differentiation of B lymphocytes and correct lymphoid atrophy and hypoplasia observed in animal models of GVHD (Piguet et al., J.Exp.Med.166:1280 (1987); Hattori et al. , Blood 90:542 (1997)).

GVHD is initiated by intravenous injection of approximately 1-5 x ¹⁰⁸ C57BL/6 splenocytes into (BALB/c X C57BL/6) F1 mice (both purchased from Jackson Lab, Bar Harbor, Maine) . After injection of parental cells, when lymphoid atrophy and hypoplasia is mild (approximately 5 days), moderate (approximately 12 days) or severe (approximately 20 days), 6-8 mice are injected daily intraperitoneally, intramuscularly Neutrokine-α or control buffer was injected intradermally or intradermally at 0.1-5.0 mg/kg. The effect of Neutrokine-α on lymphoid atrophy and hypoplasia of the spleen was analyzed by FACS and histopathology at various time points (3-4) between 10-30 days. Briefly, splenocytes were prepared from normal CBF1, GVHD, or Neutrokine-α-treated mice with fluorescein-phycoerythrin-conjugated anti-H-2Kb antibody, biotin-conjugated anti-H-2Kd antibody , and FITC-conjugated anti-CD4 antibody, anti-CD8 antibody, or anti-B220 antibody, followed by CyChrome-conjugated avidin staining. All of these antibodies are commercially available from PharMingen (San Diego, CA). Cells were then analyzed on a FACScan (Becton Dickinson, San Jose, CA). Recipient and donor lymphocytes were identified as H-2Kb+Kd+ and H-2Kb+Kd- cells, respectively. The number of recipient or donor CD4+T, CD8+T and B220+B cells was calculated from the total number of splenocytes recovered, and the percentage of each subpopulation was determined by trichrome analysis. Histological evaluation of the relative extent of tissue damage in other GVHD-associated organs (liver, skin and small intestine) was performed after sacrifice of the animals.

Finally, Neutrokine-alpha and buffer treated animals were clinically evaluated every other day for cachexia, body weight and lethality.

Agonists and antagonists of Neutrokine-α can also be tested in this mouse model of acute GVHD.

Example 9: Isolation of Anti-Neutrokine-α Polypeptide Antibody Fragments from scFvs Library

Naturally occurring V genes isolated from human PBLs were constructed into large libraries of antibody fragments containing reactivity against Neutrokine-α and/or Neutrokine-αSV with or without contact with the donor (see U.S. Patent 5,885,793, incorporated herein by reference in its entirety).

library rescue

A library of scFvs was constructed from RNA from human PBLs as described in WO92/01047 (hereby incorporated by reference in its entirety). To rescue phage-displayed antibody fragments, approximately ^{10 E.} coli carrying phagemids were used to inoculate 50 ml of 2×TY (2×TY-AMP-GLU) containing 1% glucose and 100 μg/ml ampicillin, and shaken Grow to an OD of 0.8. 5 ml of this culture was used to inoculate 50 ml of 2×TY-AMP-GLU, 2×10 ⁸ TU of the delta gene 3 helper gene (M13 delta gene III, see WO92/01047) was added, and the culture was incubated at 37° C. without shaking Incubate for 45 minutes, then incubate with shaking for 45 minutes at 37°C. The culture was centrifuged at 4000 rpm for 10 minutes and the pellet was resuspended in 2 L of 2xTY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grown overnight. Phage were prepared as described in WO92/01047.

The M13δ gene III is prepared as follows: M13δ gene III helper phage does not encode gene III protein, but the phage (particle) display antibody fragment has a strong ability to bind antigen. Infectious M13delta gene III particles are produced by growing helper phage during phage morphogenesis in cells carrying pUC19 derivatives that provide wild-type gene III protein. The culture was incubated at 37°C for 1 hour without shaking, then at 37°C for 1 hour with shaking. Invert the cells (IEC-Centra 84000 revs/min, 10 minutes), resuspend in 300ml 2×TY broth (2×TY-AMP-KAN) containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, Grow overnight at 37°C with shaking. Phage particles were purified and concentrated from the medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml of PBS, and filtered through a 0.45 μm filter (MinisartNML; Sartorius) to a final concentration of approximately ¹⁰ Transducing units/ml (ampicillin resistant clones).

panned library

Immunization tubes (Nunc) were coated overnight with 4 ml of 100 μg/ml or 10 μg/ml of the polypeptide of the present invention in PBS. The tube was blocked with 2% Marvel-PBS at 37°C for 2 hours, and then washed 3 times with PBS. About 10 ¹³ TU of phage was added to the test tube, and incubated at room temperature for 30 minutes, and then rested for 1.5 hours. The tube was washed 10 times with PBS 0.1% Tween-20, and 10 times with PBS. Add 1ml of 100mM triethylamine to elute the phage, rotate it up and down for 15 minutes, and immediately neutralize it with 0.5ml of 1.0M Tris-HCl, pH7.4. The phage were then used to infect 10 ml of mid-log phase E. coli TG1 by incubating the eluted phage with the bacteria for 30 minutes at 37°C. E. coli were plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library was then phage-panned using the delta gene 3 assisted phage described above to prepare phage for subsequent selection. This step was repeated 4 times in total, the affinity purification was increased to 20 times for washing the test tube with PBS, 0.1% Tween-20, and 20 times for washing with PBS.

Qualitative conjugates

Eluted phage from the third and fourth selections were used to infect E. coli HB2151 and produce soluble scFv from a single colony (Marks et al., 1991). ELISAs were performed on microtiter plates coated with 10 pg/ml polypeptide of the invention in 50 mM bicarbonate, pH 9.6. Positive clones in ELISA were further characterized by PCR fingerprinting (see WO92/01047) and then sequenced.

Example 10: Neutrokine-α/Neutrokine-α receptor interaction neutralization with anti-Neutrokine-α monoclonal antibody

Monoclonal antibodies against Neutrokine-α protein were generated according to the following method. Briefly, mice were injected subcutaneously with 50 μg of His-tagged Neutrokine-α produced by the method of Example 2 in 100 μl of PBS emulsified in 100 μl of complete Freunds adjuvant. 25 μg Neutrokine-α in incomplete Freunds' adjuvant was subcutaneously injected once every two weeks, for a total of three injections. Animals were rested for one month and finally 25 μg of Neutrokine-α in PBS was advanced intraperitoneally. Animals were sacrificed four days later, and splenocytes were taken for fusion.

The "fusion" procedure was performed by fusing cells from the spleen with 2 × 10E7 P3X63Ag8.653 plasmacytoma cells using PEG1500 (Boehringer Mannheim) according to the manufacturer's instructions (see Gefter, M.L. et al., Somatic Cell Genet 3: 231-36 (1977); Boehringer Mannheim PEG1500 (Cat. No. 783641), Product Description).

After confluence, cells were resuspended in 400 ml HAT medium supplemented with 20% FBS and 4% Hybridoma Supplement (Boehringer Mannheim) and distributed in 96-well plates at a density of 200 μl/well. After 7 days of fusion, 100 μl of medium was aspirated and replaced with 100 μl of fresh medium. 14 days after fusion, hybridomas were screened for antibody production.

Hybridoma supernatants were screened by ELISA for binding to Neutrokine-α protein immobilized on the plate. Plates were coated with Neutrokine-α by incubating 100 μl/well of Neutrokine-α at a concentration of 2 μg/ml in PBS overnight. Hybridoma supernatants were diluted 1:10 with PBS, placed in individual wells of Neutrokine-α-coated plates, and incubated overnight at 4°C. On the next day, the plate was washed 3 times with PBS containing 0.1% Tween-20, and the color reaction was carried out with anti-mouse IgG ABC system (Vector Laboratories). Add 25ml/well of 2M H ₂ SO ₄ to stop the color reaction. Plates were read at 450nm.

Ig isotypes of hybridoma supernatants were detected with Isostrips. Cloning was performed by limiting dilution on HT medium. Approximately 3 x 10E6 cells in 0.9 ml of HBSS were injected into pristane-primed mice. After 7-9 days, ascitic fluid was collected with a 19 g syringe. All antibodies were purified by protein G affinity chromatography using an Acta FPLC system (Pharmacia).

All three mice showed a strong immune response initially and after two consecutive subcutaneous injections; serum titers were 10E-7 as determined by ELISA on Neutrokine-alpha coated plates.

In one assay, more than 1000 primary hybridomas were generated using splenocytes from positive mice. 917 of them were screened for anti-Neutrokine-α antibody. A 1:1 dilution of the supernatant was used for screening to detect all positive clones. Of the 917 hybridomas screened, 76 were found to be positive, of which 17 were IgG producers. After affinity testing and cloning, 9 of them were selected for further expansion and purification.

All purified monoclonal antibodies were able to bind different forms of Neutrokine-α (including His-tagged and baculovirus-derived proteins (see Example 2)) in Western blot analysis and ELISA. Six of these nine clones were also able to bind Neutrokine-α on the surface of THP-1 cells. However, none of the antibodies tested were able to capture Neutrokine-alpha from solution.

Production of high-affinity anti-Neutrokine-α monoclonal antibodies that recognize Neutrokine-α expressed on the cell surface but not in solution, which can be used for neutralization studies in vivo, and in In vitro monocyte and B cell analysis. These antibodies were also used for sensitive detection of Neutrokine-α in Western blot analysis.

In one assay, more than 1000 primary hybridomas were generated using splenocytes from positive mice. 729 of them were screened for anti-Neutrokine-α antibody. The 1:10 diluted supernatant was used for screening under stringent conditions to pick the most suitable clones with higher affinity. Of the 729 hybridomas screened, 23 were positive, including 16 IgM and 7 IgG producers (4 of the latter provided a strong IgM background). In this assay, the isotype distribution of IgG hybridomas was skewed towards the IgG2 subclass. Three of the seven IgG hybridomas produced antibodies of the IgG2a subclass, two produced antibodies of the IgG2b subclass, and the remaining two were IgG1 producers.

The supernatants of all positive hybridomas generated in the second assay were tested for their ability to inhibit Neutrokine-α-mediated B cell proliferation. In the first screening assay, two hybridomas producing IgG neutralizing antibodies (these were antibodies 16C9 and 12C5) were tested. In another experiment, the IgG neutralizing activity of the hybridomas (ie 16C9 and 12C5) was determined, while the strongly neutralizing supernatants of two other hybridomas, 15C10 and 4A6, were not identified.

These 3 clones were subsequently expanded in vivo (one clone, 15C10, was also expanded in a hollow fiber system), providing affinity chromatography purified antibodies. All three of these clones were able to bind Neutrokine-α on the surface of THP-1 cells and were also able to bind (ie capture) Neutrokine-α from solution.

Specifically, an assay was performed with the anti-Neutrokine-alpha monoclonal antibody described in the second assay above to determine whether the antibody neutralized Neutrokine-alpha/Neutrokine-alpha receptor binding. Briefly, Neutrokine-alpha protein was biotinylated with EZ-linked TNHS-biotin reagent (Pierce, Rockford, IL). Biotinylated Neutrokine-α was then used to identify cell surface proteins that bind Neutrokine-α. Preliminary tests suggest that Neutrokine-alpha binds to receptors on B lymphocytes.

Anti-Neutrokine-alpha antibodies raised in the second assay above neutralized the binding of Neutrokine-alpha to the Neutrokine-alpha receptor. In a specific embodiment, anti-Neutrokine-alpha antibody 15C10 neutralizes the binding of Neutrokine-alpha to the Neutrokine-alpha receptor.

Thus, the anti-Neutrokine-α antibodies raised in the second assay above (specifically antibody 15C10) recognize and bind membrane-bound and soluble Neutrokine-α proteins and neutralize Neutrokine-α and Neutrokine-α receptors in vitro combination.

It should be clear that the invention may be practiced otherwise than as specifically described and examples hereinabove. Various modifications of the invention are possible in light of the above teachings and within the scope of the appended claims.

All publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited are hereby incorporated by reference in their entirety.

Also, in the sequence listing filed here, and in applications 09/005874, filed January 12, 1998, US60/036100, filed January 14, 1997, and PCT/US96/17957, filed October 25, 1996 All submitted sequence listings are incorporated by reference in their entirety.

sequence listing

<110> Human Genome Science Co., Ltd.

<120> Neutrokine-α and Neutrokine-α splice variants

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<141>2000-02-22

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His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile

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Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala

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Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

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Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

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Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

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Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

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Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

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Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

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Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

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Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

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<220>

<221> misc_features

<222>(452)..(453)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(458)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(461)..(462)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(466)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(469)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(471)..(472)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(474)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(478)..(481)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(496)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(498)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(504)

<223> n is equal to a, t, g, or c

<400>8

aattcggcan agnaaactgg ttactttttt atatatggtc aggttttata tactgataag 60

acctacgcca tgggacatct agttcagagg aagaaggtcc atgtctttgg ggatgaattg 120

agtctggtga ctttgtttcg atgtattcaa aatatgcctg aaacactacc caataattcc 180

tgctattcag ctggcattgc aaaactggna ggaaggagat gaactccaac ttgcaatacc 240

aggggaaaat gcacaattat cactgggatg gagatgttca cattttttgg gtgccattga 300

aactgctgtg acctncttac ancangtgct gttngctatt ttncctncct nttctntggt 360

aacctcttag gaaggaagga ttcttaactg ggaaataacc caaaaaaann ttaaangggt 420

angngnnana ngngggggnng ttnncnngnn gnnttttngg nntatnttnt nntngggnnn 480

ngtaaaaatg gggccnangg gggnttttt 509

<210>9

<211>497

<212>DNA

<213>Homo sapiens

<220>

<221> misc_features

<222>(168)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(213)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(288)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(325)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(346)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(406)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(415)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(419)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(437)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(442)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(467)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(473)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(476)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(481)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(483)..(484)

<223> n is equal to a, t, g, or c

<220>

<221> misc_features

<222>(494)

<223>n is equal to a, t, g, or c

<400>9

aattcggcac gagcaaggcc ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa 60

tctttgaacc accagctcca ggagaaggca actccagtca gaacagcaga aataagcgtg 120

ccgttcaggg tccagaagaa acagtcactc aagactgctt gcaactgntt gcagacagtg 180

aaacaccaac tatacaaaaa ggctcccttc tgntgccaca tttgggccaa ggaatggaga 240

gatttcttcg tctggaaaca ttttgccaaa ctcttcagat actctttnct ctctgggaat 300

caaaggaaaa tctctactta gattnacaca tttgttccca tgggtntctt aagttttaaa 360

aggggagtgc ccttagggagg aaaaggggat aaatattggc caaggnactg gttantttnt 420

aaatatggtc aggtttntat anctggtagg cctcgccatg ggcattnatt canggngagg 480

ncnnctttt gggntga 497

<210>10

<211>27

<212>DNA

<213>Homo sapiens

<220>

<223>Description of combined DNA/RNA molecules: x=

deoxyinosine

<400>10

gtgggatcca gcctccgggc agagctg 27

<210>11

<211>33

<212>DNA

<213>Homo sapiens

<400>11

gtgaagcttt tattacagca gtttcaatgc acc 33

<210>12

<211>26

<212>DNA

<213>Homo sapiens

<400>12

gtgtcatgag cctccgggca gagctg 26

<210>13

<211>33

<212>DNA

<213>Homo sapiens

<400>13

gtgaagcttt tattacagca gtttcaatgc acc 33

<210>14

<211>28

<212>DNA

<213>Homo sapiens

<400>14

gtgggatccc cgggcagagc tgcagggc 28

<210>15

<211>33

<212>DNA

<213>Homo sapiens

<400>15

gtgggatcct tattacagca gtttcaatgc acc 33

<210>16

<211>129

<212>DNA

<213>Homo sapiens

<400>16

gcgggatccg ccaccatgaa ctccttctcc acaagcgcct tcggtccagt tgccttctcc 60

ctggggctgc tcctggtgtt gcctgctgcc ttccctgccc cagttgtgag acaaggggac 120

ctggccagc 129

<210>17

<211>30

<212>DNA

<213>Homo sapiens

<400>17

gtgggatcct tacagcagtt tcaatgcacc 30

<210>18

<211>903

<212>DNA

<213>Homo sapiens

<220>

<221> CDS

<222>(1)..(798)

<400>18

atg gat gac tcc aca gaa agg gag cag tca cgc ctt act tct tgc ctt 48

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1 5 10 15

aag aaa aga gaa gaa atg aaa ctg aag gag tgt gtt tcc atc ctc cca 96

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

20 25 30

cgg aag gaa agc ccc tct gtc cga tcc tcc aaa gac gga aag ctg ctg 144

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Ser Lys Asp Gly Lys Leu Leu

35 40 45

gct gca acc ttg ctg ctg gca ctg ctg tct tgc tgc ctc acg gtg gtg 192

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

50 55 60

tct ttc tac cag gtg gcc gcc ctg caa ggg gac ctg gcc agc ctc cgg 240

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65 70 75 80

gca gag ctg cag ggc cac cac gcg gag aag ctg cca gca gga gca gga 288

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

85 90 95

gcc ccc aag gcc ggc ctg gag gaa gct cca gct gtc acc gcg gga ctg 336

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

100 105 110

aaa atc ttt gaa cca cca gct cca gga gaa ggc aac tcc agt cag aac 384

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

115 120 125

agc aga aat aag cgt gcc gtt cag ggt cca gaa gaa aca gga tct tac 432

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr

130 135 140

aca ttt gtt cca tgg ctt ctc agc ttt aaa agg gga agt gcc cta gaa 480

Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu

145 150 155 160

gaa aaa gag aat aaa ata ttg gtc aaa gaa act ggt tac ttt ttt ata 528

Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile

165 170 175

tat ggt cag gtt tta tat act gat aag acc tac gcc atg gga cat cta 576

Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu

180 185 190

att cag agg aag aag gtc cat gtc ttt ggg gat gaa ttg agt ctg gtg 624

Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val

195 200 205

act ttg ttt cga tgt att caa aat atg cct gaa aca cta ccc aat aat 672

Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn

210 215 220

tcc tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa ctc 720

Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu

225 230 235 240

caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga gat 768

Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp

245 250 255

gtc aca ttt ttt ggt gca ttg aaa ctg ctg tgacctactt acaccatgtc 818

Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

260 265

tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaa ctgaaaatac 878

caaaaaaaaa aaaaaaaaa aaaaa 903

<210>19

<211>266

<212>PRT

<213>Homo sapiens

<400>19

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1 5 10 15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

20 25 30

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Ser Lys Asp Gly Lys Leu Leu

35 40 45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

50 55 60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65 70 75 80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

85 90 95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

100 105 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

115 120 125

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr

130 135 140

Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu

145 150 155 160

Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile

165 170 175

Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu

180 185 190

Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val

195 200 205

Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn

210 215 220

Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu

225 230 235 240

Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp

245 250 255

Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

260 265

<210>20

<211>136

<212>PRT

<213>Homo sapiens

<400>20

His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp

1 5 10 15

Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg

20 25 30

Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val

35 40 45

Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met

50 55 60

Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe

65 70 75 80

Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser

85 90 95

Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser

100 105 110

Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly

115 120 125

Thr Phe Leu Gly Phe Val Lys Leu

130 135

<210>21

<211>462

<212>DNA

<213>Homo sapiens

<400>21

atggctgttc agggtccgga agaaaccgtt actcaggact gccttcagct gatcgcagac 60

tctgaaactc cgaccatcca gaaaggttct tacacctttg ttccttggct gctttctttc 120

aaacgtggtt ctgccctgga agagaaagaa aacaaaatcc tggttaaaga aactggttac 180

ttctttatct acggtcaggt tctttacact gataagacct acgccatggg tcacctgatt 240

cagcgtaaga aagttcacgt tttcggtgac gagctgtctc tggttactct gtttcgctgc 300

attcagaaca tgccggaaac tcttcctaac aactcctgct actctgctgg catcgcaaaa 360

ctggaagagg gtgatgaact gcagctggca attcctcgtg aaaacgcaca aatttctctg 420

gacggtgatg taaccttctt tggtgcactg aaacttctgt aa 462

<210>22

<211>1040

<212>DNA

<213>Homo sapiens

<220>

<221> CDS

<222>(1)..(468)

<400>22

cgc gtg gta gac ctc tca gct cct cct gca cca tgc ctg cct gga tgc 48

Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys

1 5 10 15

cgc cat tct caa cat gat gat aat gga atg aac ctc aga aac aga act 96

Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr

20 25 30

tac aca ttt gtt cca tgg ctt ctc agc ttt aaa aga gga aat gcc ttg 144

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu

35 40 45

gag gag aaa gag aac aaa ata gtg gtg agg caa aca ggc tat ttc ttc 192

Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe

50 55 60

atc tac agc cag gtt cta tac acg gac ccc atc ttt gct atg ggt cat 240

Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His

65 70 75 80

gtc atc cag agg aag aaa gta cac gtc ttt ggg gac gag ctg agc ctg 288

Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

85 90 95

gtg acc ctg ttc cga tgt att cag aat atg ccc aaa aca ctg ccc aac 336

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn

100 105 110

aat tcc tgc tac tcg gct ggc atc gcg agg ctg gaa gaa gga gat gag 384

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu

115 120 125

att cag ctt gca att cct cgg gag aat gca cag att tca cgc aac gga 432

Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly

130 135 140

gac gac acc ttc ttt ggt gcc cta aaa ctg ctg taa ctcacttgct 478

Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu

145 150 155

ggagtgcgtg atccccttcc ctcgtcttct ctgtacctcc gagggagaaa cagacgactg 538

gaaaaactaa aagatgggga aagccgtcag cgaaagtttt ctcgtgaccc gttgaatctg 598

atccaaacca ggaaatataa cagacagcca caaccgaagt gtgccatgtg agttatgaga 658

aacggagccc gcgctcagaa agaccggatg aggaagaccg ttttctccag tcctttgcca 718

acacgcaccg caaccttgct ttttgccttg ggtgacacat gttcagaatg cagggagatt 778

tccttgtttt gcgatttgcc atgagaagag ggcccacaac tgcaggtcac tgaagcattc 838

acgctaagtc tcaggattta ctctcccttc tcatgctaag tacaacacg ctcttttcca 898

ggtaatacta tgggatacta tggaaaggtt gtttgttttt aaatctagaa gtcttgaact 958

ggcaatagac aaaaatcctt aaaattcaa gtgtaaaata aacttaatta aaaaggttta

1018

agtgtgaaaa aaaaaaaaaa aa

1040

<210>23

<211>155

<212>PRT

<213>Homo sapiens

<400>23

Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys

1 5 10 15

Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr

20 25 30

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu

35 40 45

Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe

50 55 60

Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His

65 70 75 80

Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

85 90 95

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn

100 105 110

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu

115 120 125

Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly

130 135 140

Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu

145 150 155

<210>24

<211>26

<212>DNA

<213>Homo sapiens

<400>24

ccaccagctc caggagaagg caactc 26

<210>25

<211>19

<212>DNA

<213>Homo sapiens

<400>25

accgcgggac tgaaaatct 19

<210>26

<211>23

<212>DNA

<213>Homo sapiens

<400>26

cacgcttatt tctgctgttc tga 23

<210>27

<211>657

<212>DNA

<213>Homo sapiens

<400>27

taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagggccac 60

cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120

gctgtcaccg caggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180

cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240

ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300

ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360

gtcaaagaaa ctggttatactt ttttatatat ggtcaggttt tatacactga taagacctat 420

gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480

gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540

tcagctggca ttgcaaaact ggaagaagga gatgaacttc aacttgcaat accacgagaa 600

aatgcacaaa tatcactgga tggagatgtc aattttttg gtgccctcaa actgctg 657

<210>28

<211>219

<212>PRT

<213>Homo sapiens

<400>28

Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu

1 5 10 15

Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro

20 25 30

Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile

35 40 45

Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg

50 55 60

Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys

65 70 75 80

Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser

85 90 95

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu

100 105 110

Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe

115 120 125

Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

130 135 140

Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

145 150 155 160

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn

165 170 175

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu

180 185 190

Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly

195 200 205

Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

210 215

<210>29

<211>657

<212>DNA

<213>Homo sapiens

<400>29

taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagagccac 60

cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120

gctgtcaccg cgggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180

cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240

ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300

ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360

gtcaaagaaa ctggttatactt ttttatatat ggtcaggttt tatacactga taagacctat 420

gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480

gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540

tcagctggca ttgcaaaact ggaagaaggg gatgaacttc aacttgcaat accacgagaa 600

aatgcacaaa tatcactgga tggagatgtc aattttttg gtgccctcaa actgctg 657

<210>30

<211>219

<212>PRT

<213>Homo sapiens

<400>30

Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu

1 5 10 15

Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro

20 25 30

Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile

35 40 45

Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg

50 55 60

Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys

65 70 75 80

Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser

85 90 95

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu

100 105 110

Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe

115 120 125

Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

130 135 140

Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

145 150 155 160

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn

165 170 175

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu

180 185 190

Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly

195 200 205

Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

210 215

<210>31

<211>38

<212>DNA

<213>Homo sapiens

<400>31

ggtcgccgtt tctaacgcgg ccgttcaggg tccagaag 38

<210>32

<211>49

<212>DNA

<213>Homo sapiens

<400>32

ctggttcggc ccaaggtacc aagcttgtac cttagatctt ttctagatc 49

<210>33

<211>21

<212>DNA

<213>Homo sapiens

<400>33

ctggtagttc ttcggagtgt g 21

<210>34

<211>19

<212>DNA

<213>Homo sapiens

<400>34

cgcgttagaa acggcgacc 19

<210>35

<211>22

<212>DNA

<213>Homo sapiens

<220>

<221> misc_features

<222>(7)

<223>n is equal to deoxyinosine

<220>

<221> misc_features

<222>(12)

<223>n is equal to deoxyinosine

<220>

<221> misc_features

<222>(16)

<223>n is equal to deoxyinosine

<400>35

taccagntgg cngccntgca ag 22

<210>36

<211>22

<212>DNA

<213>Homo sapiens

<220>

<221> misc_features

<222>(3)

<223>n is equal to deoxyinosine

<220>

<221> misc_features

<222>(14)

<223>n is equal to deoxyinosine

<220>

<221> misc_features

<222>(16)..(17)

<223>n is equal to deoxyinosine

<400>36

gtnacagcag tttnanngca cc 22

<210>37

<211>866

<212>DNA

<213>Mus musculus

<400>37

atggatgagt ctgcaaagac cctgccacca ccgtgcctct gtttttgctc cgagaaagga 60

gaagatatga aagtgggata tgatcccatc actccgcaga aggagggaggg tgcctggttt 120

gggatctgca gggatggaag gctgctggct gctaccctcc tgctggccct gttgtccagc 180

agtttcacag cgatgtcctt gtaccagttg gctgccttgc aagcagacct gatgaacctg 240

cgcatggagc tgcagagcta ccgaggttca gcaacaccag ccgccgcggg tgctccagag 300

ttgaccgctg gagtcaaact cctgacaccg gcagctcctc gaccccacaa ctccagccgc 360

ggccacagga acagacgcgc cttccaggga ccagaggaaa cagaacaaga tgtagacctc 420

tcagctcctc ctgcaccatg cctgcctgga tgccgccatt ctcaacatga tgataatgga 480

atgaacctca gaaacatcat tcaagactgt ctgcagctga ttgcagacag cgacacgccg 540

gccttggagg agaaagagaa caaaatagtg gtgaggcaaa caggctattt cttcatctac 600

agccaggtc tatacacgga ccccatcttt gctatgggtc atgtcatcca gaggaagaaa 660

gtacacgtct ttggggacga gctgagcctg gtgaccctgt tccgatgtat tcagaatatg 720

cccaaaacac tgcccaacaa ttcctgctac tcggctggca tcgcgaggct ggaagaagga 780

gatgagattc agcttgcaat tcctcgggag aatgcacaga tttcacgcaa cggagacgac 840

accttctttg gtgccctaaa actgct 866

<210>38

<211>177

<212> DNA

<213>Mus musculus

<400>38

mdsaktcccs kgdmkvgydt kgawgcrdgr aatassstam syaaadmnrm syrgsataaa 60

gatagvktaa rhnssrghrn rragtdvdsa acgcrhshdd ngmnrndcad sdtaknkvvr 120

tgyysvytda mghvrkkvhv gdsvtrcnmk tnnscysaga rgdarnasrn gddtgak 177

Form PCT/RO/134

Instructions on preservation of microorganisms

(PCT Rule 13bis)

Form PCT/RO/134 (July 1992)

ATCC Deposit No. 97768

Canada

The applicant requests that the Commissioner of Patents authorize the supply of samples of deposited biological material referred to in an application only when a Canadian patent based on the application is granted or the application is refused or abandoned or not reinstated or withdrawn An independent expert designated by the Director, the applicant must notify the International Bureau in a written statement before the technical preparations for publication of the international application are completed.

Norway

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Norwegian Patent Office) or if the Norwegian Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Norwegian Patent Office no later than the time of publication under Sections 22 and 33(3) of the Norwegian Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Norwegian Patent Office or anyone agreed by the applicant in the individual case.

Australia

Applicants hereby point out that microbiological samples are only available to skilled persons who have no interest in the invention prior to the grant of the patent or before the patent application is abandoned, refused or withdrawn (section 3.25(3) of the Australian Patents Act).

Finland

The applicant hereby requests that samples be made available to experts in the field only when the application is published (by the State Patent Office) or when the State Patent Office ultimately decides not to publish it.

united kingdom

Applicant hereby requests that microbiological samples be provided only to specialists. The applicant must notify the International Bureau of this request in a written statement before the technical preparations for publication of the international application have been completed.

Denmark

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Danish Patent Office) or if the Danish Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Danish Patent Office no later than the time of publication under Sections 22 and 33(3) of the Danish Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Danish Patent Office or anyone agreed by the applicant in the individual case.

Sweden

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Swedish Patent Office) or if the Swedish Patent Office ultimately decides not to publish it. The request should be lodged with the International Bureau (preferably on form PCT/RO/134 in Annex Z of Volume I of the PCT Applicant's Guide) within 16 months from the priority date. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Swedish Patent Office or anyone agreed by the applicant in the individual case.

Netherlands

The applicant requests that a sample be made available to the expert under section 31F(1) of the Patents Act only when the Dutch patent is granted or if the application is refused or withdrawn or invalidated. The request must be lodged by the applicant with the Netherlands Industrial Property Office before publication under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever is earlier.

Instructions on preservation of microorganisms

(PCT Rule 13bis)

Form PCT/RO/134 (July 1992)

ATCC Deposit No. 203518

Canada

The applicant requests that the Commissioner of Patents authorize the supply of samples of deposited biological material referred to in an application only when a Canadian patent based on the application is granted or the application is refused or abandoned or not reinstated or withdrawn An independent expert designated by the Director, the applicant must notify the International Bureau in a written statement before the technical preparations for publication of the international application are completed.

Norway

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Norwegian Patent Office) or if the Norwegian Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Norwegian Patent Office no later than the time of publication under Sections 22 and 33(3) of the Norwegian Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Norwegian Patent Office or anyone agreed by the applicant in the individual case.

Australia

Applicants hereby point out that microbiological samples are only available to skilled persons who have no interest in the invention prior to the grant of the patent or before the patent application is abandoned, refused or withdrawn (section 3.25(3) of the Australian Patents Act).

Finland

The applicant hereby requests that samples be made available to experts in the field only when the application is published (by the State Patent Office) or when the State Patent Office ultimately decides not to publish it.

united kingdom

Applicant hereby requests that microbiological samples be provided only to specialists. The applicant must notify the International Bureau of this request in a written statement before the technical preparations for publication of the international application have been completed.

Denmark

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Danish Patent Office) or if the Danish Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Danish Patent Office no later than the time of publication under Sections 22 and 33(3) of the Danish Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Danish Patent Office or anyone agreed by the applicant in the individual case.

Sweden

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Swedish Patent Office) or if the Swedish Patent Office ultimately decides not to publish it. The request should be lodged with the International Bureau (preferably on form PCT/RO/134 in Annex Z of Volume I of the PCT Applicant's Guide) within 16 months from the priority date. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Swedish Patent Office or anyone agreed by the applicant in the individual case.

Netherlands

The applicant requests that a sample be made available to the expert under section 31F(1) of the Patents Act only when the Dutch patent is granted or if the application is refused or withdrawn or invalidated. The request must be lodged by the applicant with the Netherlands Industrial Property Office before publication under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever is earlier.

Instructions on the preservation of microorganisms

(PCT Rule 13bis)

Form PCT/RO/134 (July 1992)

ATCC Deposit No. PTA-1158

Canada

The applicant requests that the Commissioner of Patents authorize the supply of samples of deposited biological material referred to in an application only when a Canadian patent based on the application is granted or the application is refused or abandoned or not reinstated or withdrawn An independent expert designated by the Director, the applicant must notify the International Bureau in a written statement before the technical preparations for publication of the international application are completed.

Norway

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Norwegian Patent Office) or if the Norwegian Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Norwegian Patent Office no later than the time of publication under Sections 22 and 33(3) of the Norwegian Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Norwegian Patent Office or anyone agreed by the applicant in the individual case.

Australia

Applicants hereby point out that microbiological samples are only available to skilled persons who have no interest in the invention prior to the grant of the patent or before the patent application is abandoned, refused or withdrawn (section 3.25(3) of the Australian Patents Act).

Finland

The applicant hereby requests that samples be made available to experts in the field only when the application is published (by the State Patent Office) or when the State Patent Office ultimately decides not to publish it.

united kingdom

Applicant hereby requests that microbiological samples be provided only to specialists. The applicant must notify the International Bureau of this request in a written statement before the technical preparations for publication of the international application have been completed.

Denmark

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Danish Patent Office) or if the Danish Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Danish Patent Office no later than the time of publication under Sections 22 and 33(3) of the Danish Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Danish Patent Office or anyone agreed by the applicant in the individual case.

Sweden

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Swedish Patent Office) or if the Swedish Patent Office ultimately decides not to publish it. The request should be lodged with the International Bureau (preferably on form PCT/RO/134 in Annex Z of Volume I of the PCT Applicant's Guide) within 16 months from the priority date. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Swedish Patent Office or anyone agreed by the applicant in the individual case.

Netherlands

The applicant requests that a sample be made available to the expert under section 31F(1) of the Patents Act only when the Dutch patent is granted or if the application is refused or withdrawn or invalidated. The request must be lodged by the applicant with the Netherlands Industrial Property Office before publication under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever is earlier.

Instructions on the preservation of microorganisms

(PCT Rule 13bis)

Form PCT/RO/134 (July 1992)

ATCC Deposit No. PTA-1159

Canada

The applicant requests that the Commissioner of Patents authorize the supply of samples of deposited biological material referred to in an application only when a Canadian patent based on the application is granted or the application is refused or abandoned or not reinstated or withdrawn An independent expert designated by the Director, the applicant must notify the International Bureau in a written statement before the technical preparations for publication of the international application are completed.

Norway

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Norwegian Patent Office) or if the Norwegian Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Norwegian Patent Office no later than the time of publication under Sections 22 and 33(3) of the Norwegian Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Norwegian Patent Office or anyone agreed by the applicant in the individual case.

Australia

Applicants hereby point out that microbiological samples are only available to skilled persons who have no interest in the invention prior to the grant of the patent or before the patent application is abandoned, refused or withdrawn (section 3.25(3) of the Australian Patents Act).

Finland

The applicant hereby requests that samples be made available to experts in the field only when the application is published (by the State Patent Office) or when the State Patent Office ultimately decides not to publish it.

united kingdom

Applicant hereby requests that microbiological samples be provided only to specialists. The applicant must notify the International Bureau of this request in a written statement before the technical preparations for publication of the international application have been completed.

Denmark

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Danish Patent Office) or if the Danish Patent Office ultimately decides not to publish it. The request shall be submitted by the applicant to the Danish Patent Office no later than the time of publication under Sections 22 and 33(3) of the Danish Patent Act. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Danish Patent Office or anyone agreed by the applicant in the individual case.

Sweden

The applicant hereby requests that samples be made available to experts in the field only if the application is published (by the Swedish Patent Office) or if the Swedish Patent Office ultimately decides not to publish it. The request should be lodged with the International Bureau (preferably on form PCT/RO/134 in Annex Z of Volume I of the PCT Applicant's Guide) within 16 months from the priority date. If the applicant submits such a request, requests for samples from third parties shall specify the experts used. An expert can be anyone on the list of experts recognized by the Swedish Patent Office or anyone agreed by the applicant in the individual case.

Netherlands

The applicant requests that a sample be made available to the expert under section 31F(1) of the Patents Act only when the Dutch patent is granted or if the application is refused or withdrawn or invalidated. The request must be lodged by the applicant with the Netherlands Industrial Property Office before publication under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever is earlier.

Claims (23)

1. by the hybridoma excretory antibody of ATCC preserving number PTA-1159 or PTA-1158.

2. comprise the variable region of heavy chain (VH) of the antibody of claim 1 and the antibody of variable region of light chain (VL).

3. comprise all the VH CDR of antibody of claim 1 and the antibody of VL CDR, it combines with proteinic film combining form or the soluble form specificity of SEQ ID NO:2.

4. the antibody of claim 2, it is selected from as next group:

(a) monoclonal antibody;

(b) chimeric antibody;

(c) humanized antibodies;

(d) people's antibody;

(e) single-chain antibody;

(f) Fab fragment; With

(g) F (ab ') 2 fragments.

5. the antibody of claim 3, it is selected from as next group:

(a) monoclonal antibody;

(b) chimeric antibody;

(c) humanized antibodies;

(d) people's antibody;

(e) single-chain antibody;

(f) Fab fragment; With

(g) F (ab ') 2 fragments.

6. each antibody of claim 1-5, it is a mark.

7. the antibody of claim 6, wherein said mark is selected from as next group:

(a) enzyme labelling;

(b) radio isotope;

(c) fluorescent mark; With

(d) vitamin H.

8. the antibody of claim 7, wherein said mark is a kind of radio isotope that is selected from as next group:

(a) ¹²⁵I；

(b) ¹²¹I；

(c) ¹³¹I；

(d) ¹¹²In; With

(e) ⁹⁹Tc。

9. each antibody of claim 1-5, wherein said antibody is puted together with a therapeutical agent or cytotoxic agent.

10. the antibody of claim 9, wherein said therapeutical agent or cytotoxic agent are selected from as next group:

(a) metabolic antagonist;

(b) alkylating agent;

(c) microbiotic;

(d) somatomedin;

(e) cytokine;

(f) anti-angiogenic agent;

(g) antimitotic agent;

(h) anthracycline antibiotics;

(i) toxin; With

(j) apoptosis agent.

11. comprise each the pharmaceutical composition of antibody of claim 1-10.

12. comprise each the diagnosis composition of antibody of claim 1-10.

13. produce each the isolated cells of antibody of claim 1-5.

14. produce the hybridoma of (a) described monoclonal antibody in the claim 5.

15.ATCC the hybridoma of preserving number PTA-1159 or PTA-1158.

The proteic method of Neutrokine-α 16. detection of biological imitates in the product comprises:

(a) each antibody of described biological sample and claim 1-10 is contacted; With

(b) detect Neutrokine-α albumen.

17. each antibody of claim 1-10 is used for the treatment of, prevents in preparation or diagnoses disease of immune system or disorderly pharmaceutical composition or the purposes in the diagnosis composition.

18. the purposes of claim 17, wherein said disease of immune system or disorder are selected from as next group:

(a) inflammatory diseases or disorder;

(b) leukemia;

(c) tumour;

(d) metastases; With

(e) lymphadenopathy.

19. each antibody of claim 1-10 is used for the treatment of, prevents in preparation or diagnoses the pharmaceutical composition of graft versus host disease or the purposes in the diagnosis composition.

20. each antibody of claim 1-10 is used for the treatment of, prevents in preparation or diagnoses autoimmunization systemic disease or disorderly pharmaceutical composition or the purposes in the diagnosis composition.

21. the purposes of claim 20, wherein said autoimmunization systemic disease or disorder are selected from as next group:

(a) rheumatoid arthritis;

(b) systemic lupus erythematous;

(c) multiple sclerosis;

(d) Sjogren ' s syndrome;

(e) IgA nephropathy;

(f) glomerulonephritis;

(g) diabetes; With

(h) myasthenia gravis.

22. each antibody of claim 1-10 is used for the treatment of, prevents in preparation or diagnoses the pharmaceutical composition of immune deficiency or the purposes in the diagnosis composition.

23. the purposes of claim 22, wherein said immune deficiency are selected from as next group:

(a) common mutability immune deficiency (CVID);

(b) acquired immune deficiency syndrome (AIDS) (AIDS);

(c) severe severe combined immunodeficiency (SCID)

(d) gamma-globulin is very few in the blood; With

(e) Wiskott-Aldrich syndrome.

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