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WO2000050459A1 - Proteines tr11, tr11sv1, et tr11sv2 du type recepteur tnf humain - Google Patents

Proteines tr11, tr11sv1, et tr11sv2 du type recepteur tnf humain Download PDF

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Publication number
WO2000050459A1
WO2000050459A1 PCT/US2000/004572 US0004572W WO0050459A1 WO 2000050459 A1 WO2000050459 A1 WO 2000050459A1 US 0004572 W US0004572 W US 0004572W WO 0050459 A1 WO0050459 A1 WO 0050459A1
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trl
seq
polypeptide
amino acid
acid residues
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PCT/US2000/004572
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English (en)
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Steven M. Ruben
Jian Ni
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Human Genome Sciences, Inc.
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Priority to AU38601/00A priority Critical patent/AU3860100A/en
Publication of WO2000050459A1 publication Critical patent/WO2000050459A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TRll Human Tumor Necrosis Factor Receptor-Like Proteins TRll, TRllSVl, and TR11SV2
  • TR11 receptor human TNF receptor-related protein
  • TRl lSVl and TR11SV2 receptors two splice variants thereof, referred to herein as the TRl lSVl and TR11SV2 receptors, of Figures 2A and 2B and 3A and 3B, respectively, each having considerable homology to murine glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR).
  • TR11, TR11SV1, and TR11SV2 polypeptides are also provided.
  • vectors, host cells and recombinant methods for producing the same The invention also relates to both the inhibition and enhancement of the activities of TRl l, TRl lSVl, and TRHSV2 receptor polypeptides and diagnostic methods for detecting TR11 receptor gene expression.
  • TNF-alpha and beta are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol, 7:625-655 (1989)).
  • Tumor necrosis factor (TNF-alpha and TNF-beta) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine playing important roles in a host of biological processes and pathologies.
  • TNF-alpha TNF-alpha
  • TNF-beta lymphotoxin-alpha
  • LT-beta LT-beta
  • TRAIL ligands for the Fas receptor
  • CD30, CD27, CD40 (also known as CDw40), OX40 and 4- IBB receptors CD30, CD27, CD40 (also known as CDw40), OX40 and 4- IBB receptors.
  • TNF-beta Both TNF-alpha and TNF-beta function as homotrimers when they bind to TNF receptors.
  • TNF is produced by a number of cell types, including monocytes, fibroblasts, T-cells, natural killer (NK) cells and predominately by activated macrophages.
  • TNF- alpha has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata, et al, J. Immunol. 136:2483 (1987)), growth regulation, vascular endothelium effects and metabolic effects.
  • TNF-alpha also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation.
  • TNF-alpha up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1.
  • TNF-alpha and the Fas ligand have also been shown to induce programmed cell death.
  • TNF-beta has many activities, including induction of an antiviral state and tumor necrosis, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle, N. and Homer, R., Prog. Allergy 40:162-182 (1988)).
  • TNF-alpha and TNF-beta are involved in growth regulation and interact with hemopoietic cells at several stages of differentiation, inhibiting proliferation of various types of precursor cells, and inducing proliferation of immature myelomonocytic cells (Porter, A., Tibtech 9:158-162 (1991)).
  • mice deficient in TNF- beta production show abnormal development of the peripheral lymphoid organs and morphological changes in spleen architecture (reviewed by Aggarwal, et al, Eur Cytokine Netw, 7:93-124 (1996)).
  • the lymphoid organs the popliteal, inguinal, para-aortic, mesenteric, axillary and cervical lymph nodes failed to develop in TNF-beta -/- mice.
  • peripheral blood from TNF-beta -/- mice contained a three fold reduction in white blood cells as compared to normal mice.
  • TNF-beta -/- mice Peripheral blood from TNF-beta -/- mice, however, contained four fold more B cells as compared to their normal counterparts. Further, TNF-beta, in contrast to TNF-alpha, has been shown to induce proliferation of EB V-infected B cells. These results indicate that TNF-beta is involved in lymphocyte development.
  • TNF-alpha or TNF-beta The first step in the induction of the various cellular responses mediated by TNF-alpha or TNF-beta is their binding to specific cell surface or soluble receptors.
  • TNF-RI Two distinct TNF receptors of approximately 55-KDa
  • 75-KDa TNF- RII
  • human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, et al, Cell, 61:351 (1990)).
  • Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions.
  • TNF-RI and TNF-RII share 28% identity and are characterized by four repeated cysteine-rich motifs with significant intersubunit sequence homology.
  • the majority of cell types and tissues appear to express both TNF receptors and both receptors are active in signal transduction, however, they are able to mediate distinct cellular responses. Further, TNF-RII was shown to exclusively mediate human T-cell proliferation by TNF as shown in PCT WO 94/09137.
  • TNF-RI dependent responses include accumulation of C-FOS, IL-6, and manganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2 receptor and MHC class I and II cell surface antigen expression, growth inhibition, and cytotoxicity.
  • TNF-RI also triggers second messenger systems such as phospholipase A, protein kinase C, phosphatidylcholine-specific phospholipase C and sphingomyelinase (Pfefferk, et al, Cell, 73:457-467 (1993)).
  • phospholipase A protein kinase C
  • phosphatidylcholine-specific phospholipase C sphingomyelinase
  • Retinoic acid for example, has been shown to induce the production of TNF receptors in some cells type while down regulating production in other cells.
  • TNF-alpha has been shown to affect the localization of both types of receptor. TNF-alpha induces internalization of TNF-RI and secretion of TNF- RII (reviewed in Aggarwal, et al, supra). Thus, the production and localization of both TNF-Rs are regulated by a variety of agents.
  • the present invention provides isolated nucleic acid molecules comprising or alternatively consisting of, polynucleotides encoding TR11 , TR11SV 1, and TR11SV2 receptors having the amino acid sequences shown in Figures 1A and IB (SEQ ID NO:2), 2A and 2B (SEQ ID NO:4), and 3A and 3B (SEQ ID NO:6), respectively, or the amino acid sequences encoded by the cDNA clones encoding the TRl l, TRl lSVl, and TR11SV2 receptors, respectively, deposited as ATCC Deposit Numbers 209341, 209342, and 209343, respectively, on October 7, 1997.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR11, TR11SV1, and TR11SV2 polypeptides or peptides by recombinant techniques.
  • TRll, TRl lSVl, and/or TR11SV2 polypeptides of the invention are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), DiGeorge anomaly, Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, hypogammaglobulinemia
  • an immunodeficiency e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
  • TR11, TR11SV1, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency.
  • TRl l, TRllSVl, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-linked agammaglobulinemia.
  • TRll, TRllSVl, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose severe combined immunodeficiency (SCID).
  • SCID severe combined immunodeficiency
  • TR 11 , TR 11 S V 1 , and/or TR 11 S V2 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose Wiskott-Aldrich syndrome.
  • TRll, TRl lSVl, and/or TR11SV2 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.
  • TRll, TRllSVl, and/or TR11SV2 antagonists are administered to treat, prevent, prognose and/or diagnose an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein
  • autoimmune thyroiditis i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease , (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura , schleroderma 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 pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active he
  • hypothyroidism i.e., Hashimoto's thyroiditis
  • rheumatoid arthritis is treated, prevented, prognosed and/or diagnosed using anti-TRl l, TRl lSVl, and/or TR11SV2 antibodies and/or other antagonist of the invention.
  • systemic lupus erythemosus is treated, prevented, prognosed, and/or diagnosed using anti-TRl l, TRllSVl, and/or TRl 1SV2 antibodies and/or other antagonist of the invention.
  • idiopathic thrombocytopenia purpura is treated, prevented, prognosed, and/or diagnosed using anti-TRl 1, TRl ISVl, and/or TRl 1SV2 antibodies and/or other antagonist of the invention.
  • IgA nephropathy is treated, prevented, prognosed and/or diagnosed using anti-TRl 1, TRl ISVl, and/or TRl 1SV2 antibodies and/or other antagonist of the invention.
  • the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, prognosed and/or diagnosed using anti- TRl l, TRllSVl, and/or TRl 1SV2 antibodies.
  • compositions comprising a TRl 1, TRl ISVl, and/or TRl 1SV2 polynucleotide, a TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptide, and/or an anti-TRl 1, TRl ISVl, and/or TRl 1SV2 antibody, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism.
  • the compositions of the invention comprise a TRl 1 ,
  • compositions of the invention comprise a TRl 1, TRl ISVl, and/or TRl 1SV2 polynucleotide for expression of a TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptide in a host organism for treatment of disease.
  • the compositions of the invention comprise a TRl 1, TRl ISVl, and/or TRl 1SV2 polynucleotide for expression of a TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency.
  • TRll, TRl lSVl, and/or TR11SV2 gene e.g., expression to enhance the normal B-cell function by expanding B-cell numbers or increasing B cell lifespan; or expression to enhance the normal T cell function by expanding T cell numbers or increasing T cell lifespan).
  • the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by TRl 1 , TRllSVl, and TR11SV2 receptors, which involves contacting cells which express TRl 1, TRl ISVl or TRl 1SV2 receptors with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
  • a screening assay for agonists and antagonists involves determining the effect a candidate compound has on the binding of cellular ligands to TRll, TRl ISVl, and TRHSV2 receptors.
  • the method involves contacting TRl l, TRl lSVl, and TRl 1SV2 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TRll, TRllSVl, and TR11SV2 receptors is increased or decreased due to the presence of the candidate compound.
  • the invention further provides a diagnostic method useful during diagnosis or prognosis of one or more disease states resulting from aberrant cell proliferation due to alterations in TRl 1, TRl ISVl, and TRl 1SV2 receptor expression.
  • An additional aspect of the invention is related to a method for treating, detecting, and/or preventing an individual in need of an increased level of a TRl 1, TRl ISVl or TRl 1SV2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of isolated TRl 1, TRl ISVl or TRl 1SV2 polypeptides of the invention or an agonist thereof.
  • a still further aspect of the invention is related to a method for treating, detecting, and/or preventing an individual in need of a decreased level of a TRl 1,
  • TRllSVl or TR11SV2 receptor activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a TRl 1, TRl ISVl or TRl 1SV2 receptor antagonist.
  • the invention additionally provides soluble forms of the polypeptides of the present invention.
  • Soluble peptides are defined by amino acid sequences wherein the sequence comprises the polypeptide sequence lacking a transmembrane domain.
  • Such soluble forms of the TRl 1, TRl ISVl, and TRl 1SV2 receptors are useful as antagonists of the membrane bound forms of the receptors.
  • Figures 1A and IB show the nucleotide (SEQ ID NO:l) and deduced amino acid (SEQ ID NO:2) sequence of a TRl 1 receptor.
  • a potential secretory leader sequence has been predicted for the complete polypeptide, of about 25 amino acid residues.
  • the predicted secretory leader sequence is underlined in Figures 1A and IB (amino acid residues -25 to -1 in SEQ ID NO:2).
  • the deduced complete amino acid sequence includes 234 amino acid residues and has a deduced molecular weight of about 25,113 Da.
  • amino acid residues from about 26 to about 162 in Figures 1A and IB (amino acid residues 1 to 137 in SEQ ID NO:2) constitute the extracellular domain; from about 163 to about 179 (amino acid residues 138 to 154 in SEQ ID NO:2) constitute the transmembrane domain; and from about 180 to about 234 (amino acid residues 155 to 209 in SEQ LD NO:2) constitute the intracellular domain.
  • Figures 2A and 2B shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequence of a TRl ISVl receptor.
  • the deduced complete amino acid sequence includes 241 amino acid residues and has a deduced molecular weight of about 26,029 Da. It is further predicted that amino acid residues from about 1 to about 162 in Figures 2A and 2B (amino acid residues 1 to 162 in SEQ ID NO:4) constitute the extracellular domain; from about 163 to about 179 (amino acid residues
  • transmembrane domain 163 to 179 in SEQ ID NO:4) the transmembrane domain; and from about 180 to about 241 (amino acid residues 180 to 241 in SEQ ID NO:4) the intracellular domain.
  • Figures 3A and 3B shows the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequence of a TRl 1SV2 receptor.
  • a potential secretory leader sequence has been predicted for the complete polypeptide, of about 19 amino acid residues.
  • the predicted secretory leader sequence is underlined in Figures 3A and 3B (amino acid residues -19 to -1 in SEQ ED NO:6).
  • the deduced complete amino acid sequence includes 240 amino acid residues and has a deduced molecular weight of about 25,727 Da.
  • amino acid residues from about 20 to about 168 in Figures 3A and 3B constitute the extracellular domain; from about 169 to about 185 (amino acid residues 150 to 166 in SEQ ID NO:6) the transmembrane domain; and from about 186 to about 240 (amino acid residues 167 to 221 in SEQ ID NO:6) the intracellular domain.
  • a single potential asparagine-linked glycosylation site is marked in the amino acid sequence of TRl 1, TRl ISVl, and TRl 1SV2.
  • the potential site of glycosylation is at asparagine-146 in Figures 1A and IB (asparagine-121 in SEQ ID NO:2), asparagine-146 in Figure 2A and 2B (asparagine-146 in SEQ ID NO:4), and asparagine-152 in Figures 3A and 3B (asparagine-133 in SEQ ID NO:6).
  • the potential glycosylation sites are marked with a bold pound symbol (#) above the nucleotide sequence coupled with a bolded one letter abbreviation for the asparagine (N) in the amino acid sequence in Figures 1A and IB, 2 A and 2B, and 3 A and 3B.
  • Regions of high identity between TRl 1, TRl ISVl, and TRl 1SV2 and the closely related murine GITR are delineated in Figures 1A and IB, 2A and 2B, and 3A and 3B with a double underline. These regions are not limiting and are labeled as conserveed Domain
  • Conserved Domain (CD)-I is found only in TRllSVl and TRl 1SV2 (i.e., Figures 2A and 2B and 3A and 3B) and CD-VIII is found only in TRllSVl (i.e., Figures 2A and 2B).
  • Figures 4A and 4B show an alignment of the amino acid sequences of the murine glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) receptor-like molecule, TRll, TRl lSVl, and TRHSV2 (SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively).
  • the numbering of the TRll amino acid sequences shown in this figure are relative to that presented in Figures 1A and IB, 2A and 2B, and 3A and 3B, respectively.
  • the alignment was generated using the "MegAlign" module of the DNA*Star Sequence Analysis computer program (DNASTAR, Inc.).
  • mGITR Amino acid residues of mGITR, TRl ISVl, and TRl 1SV2 which do not have identity with those of TRl 1 are highlighted in black in the alignment.
  • GenBank Accession No. for mGITR is U82534 (Nocentini, G., et al, Circ. Proc. Natl. Acad. Sci. USA 94:6216-6221 (1997)).
  • Figures 5, 6, and 7 show structural analyses of the TRl l, TRl lSVl, and TRl 1SV2 receptor amino acid sequences of Figures 1 A and IB, 2A and 2B, and 3 A and 3B, respectively.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • the DNA*STAR computer program will also represent the identical data presented in Figures 5, 6, and 7 in a tabular format. Such a tabular format may assist one practicing one or more aspects of the invention in which specific structural or other features of the invention are delineated according to the data presented in Figures 5, 6, and 7 herein.
  • Such structural or other features of the polypeptides of the invention or of polynucleotides encoding such polypeptides which may be identified from the data presented in Figures 5, 6, and/or 7, or from tabular representations routinely generated from the identical data using the DNA*STAR computer program set on default settings, include, but are not limited to, Alpha, Regions - Garnier-Robson; Alpha, Regions - Chou-Fasman; Beta, Regions - Garnier-Robson; Beta, Regions - Chou-Fasman; Turn, Regions - Garnier-Robson; Turn, Regions - Chou-Fasman; Coil, Regions - Garnier- Robson; Hydrophilicity Plot - Kyte-Doolittle; Alpha, Amphipathic Regions - Eisenberg; Beta, Amphipathic Regions - Eisenberg; Flexible Regions - Karplus-Schulz; Antigenic Index - Jameson-Wolf; and Surface Probability Plot - Emini.
  • the present invention provides isolated nucleic acid molecules comprising polynucleotides encoding TRll, TRllSVl, and TR11SV2 polypeptides ( Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively), the amino acid sequences of which were determined by sequencing cloned cDNAs.
  • nucleotide sequences shown in Figures 1A and IB, 2A and 2B, and 3A and 3B were obtained by sequencing cDNA clones (Clone ID HHEAC71, HCFAZ22, and HT5EA78, respectively) containing the same amino acid coding sequences as the clones in ATCC Accession Nos. 209341, 209342, and 209343, respectively.
  • the deposited clone encoding TRl 1 is contained in the pCMVSport3.0 plasmid (Life Technologies, Rockville, MD).
  • the deposited clone encoding TRl ISVl is contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).
  • the deposited clone encoding TRl 1SV2 is contained in the pSportl plasmid (Life Technologies, Rockville, MD).
  • TRl l protein refers to all proteins resulting from the alternate splicing of the genomic DNA sequences encoding proteins having regions of amino acid sequence identity and receptor activity which correspond to the proteins shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively).
  • TRl 1 , TRl ISVl , and TRl 1SV2 proteins shown in Figures 1A and IB, 2A and 2B, and 3 A and 3B are examples of such receptor proteins.
  • nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • the information provided herein such as the nucleotide sequence in
  • nucleic acid molecules of the present invention encoding TRl 1 , TRl ISVl , and TRl 1SV2 polypeptides may be obtained using standard cloning and screening procedures, such as those used for cloning cDNAs using mRNA as starting material.
  • the nucleic acid molecule described in Figures 1A and IB (SEQ ID NO:l) was discovered in a cDNA library derived from T-helper cells.
  • a cDNA clone encoding the TRl 1 polypeptide shown in Figure 1A was not found in any other cDNA libraries examined.
  • nucleic acid molecule described in Figures 2A and 2B (SEQ ID NO:3) was discovered in a cDNA library derived from T-cells stimulated with PHA for 16 hours. A cDNA clone encoding the TRl ISVl polypeptide shown in Figure 2A and 2B was not found in any other cDNA libraries examined. Finally, the nucleic acid molecule described in Figures 3A and 3B (SEQ ID NO:5) was discovered in a cDNA library derived from activated T-cells. A cDNA clone encoding the TRl 1SV2 polypeptide shown in Figure 3A and 3B was not found in any other cDNA libraries examined. The determined nucleotide sequence of the TRl 1 cDNA of Figures 1 A and IB
  • SEQ ID NO:l contains an open reading frame encoding a protein of about 241 amino acid residues, with a single potential predicted leader sequence of about 25 amino acid residues, and a deduced molecular weight of about 25,113 Da.
  • the amino acid sequence of the potential predicted mature TRl l receptor is shown in Figures 1A and IB, from amino acid residue about 26 to residue about 234 (amino acid residues 1 to 209 in SEQ ID NO:2).
  • the TRl l protein shown in Figures 1A and IB (SEQ ID NO:2) is about 58.6% identical and about 74.1% similar to the murine mGITR receptor protein shown in SEQ ID NO:7 (see Figures 4A and 4B) using the computer program "Bestfit".
  • TRl 1 protein shown in Figures 2A and 2B contains an open reading frame encoding a protein of about 241 amino acid residues, with a deduced molecular weight of about 26,029 Da.
  • the TRl 1 protein shown in Figures 2A and 2B (SEQ ID NO:4) is about 53.1% identical and about 67.5% similar to the murine GITR receptor protein shown in SEQ ID NO:7 (see Figures 4A and 4B) using the computer program "Bestfit".
  • the determined nucleotide sequence of the TRl 1SV2 cDNA of Figure 3 A and 3B contains an open reading frame encoding a protein of about 240 amino acid residues, with a single potential predicted leader sequence of about 19 amino acid residues, and a deduced molecular weight of about 25,727 Da.
  • the amino acid sequence of the potential predicted mature TRl 1SV2 receptor is shown in Figures 3 A and 3B, from amino acid residue about 20 to residue about 240 (amino acid residues 1 to 221 in SEQ ID NO:6).
  • TRl 1SV2 protein shown in Figures 3A and 3B (SEQ ID NO:6) is about 58.6% identical and about 74.1% similar to the murine GITR receptor protein shown in SEQ ID NO:7 (see Figures 4A and 4B) using the computer program "Bestfit".
  • GITR is a 228 amino acid type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and is similar to CD27 and 4-lBB in the intracellular domain. GITR specifically protects T-cell receptor-induced apoptosis, although other apoptotic signals, including Fas triggering, dexamethasone treatment, or UN irradiation, do not. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family and appears to be involved in the regulation of T-cell receptor-mediated cell death ( ⁇ ocentini G, et al, Proc. Natl. Acad. Sci. USA
  • TRl 1 may also be involved in the regulation of cell-type specific receptor-mediated cell growth, differentiation, and, ultimately, cell death.
  • TRl 1 and TRl 1SV2 may also be involved in the regulation of cell-type specific receptor-mediated cell growth, differentiation, and, ultimately, cell death.
  • the present invention also provides mature forms of the TRl 1 and
  • TRl 1SV2 receptors of the present invention According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • the present invention provides nucleotide sequences encoding mature TRl 1 and TRl 1SV2 polypeptides having the amino acid sequences encoded by the cD ⁇ A clones contained in ATCC Deposit Numbers 209341 and 209343 and as shown in Figures 1A and IB and 3A and 3B, respectively (SEQ ID NO:2 and SEQ ID NO:6, respectively).
  • the mature TRl 1 and TRl 1SV2 polypeptides having the amino acid sequences encoded by "the cDNA clones contained in ATCC Deposit Numbers 209341 and 209343" is meant the mature form(s) of the TRl 1 and TRl 1SV2 receptors produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the deposited clones.
  • a mammalian cell e.g., COS cells, as described below
  • TRl lSVl, and TR11SV2 polypeptides shown in Figures 1A and IB, 2A and 2B, and 3A and 3B were analyzed by a computer program ("PSORT") (K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence.
  • PSORT computer program
  • the analysis by the PSORT program predicted a signal peptide cleavage site between amino acids 25 and 26 in Figures 1A and IB (-1 and +1 in SEQ ID NO:2).
  • the potential leader sequence for the TRl 1 protein shown in SEQ ID NO:2 is predicted to consist of amino acid residues -25 to -1 in SEQ ID NO:2, while the predicted mature TRl 1 protein consists of amino acid residues 1 to 209 for the TRl 1 protein shown in SEQ ID NO:2.
  • the analysis by the PSORT program predicted no signal peptide cleavage sites for the TRl ISVl protein shown in SEQ ID NO:4.
  • the analysis by the PSORT program predicted a single signal peptide cleavage site between amino acids 19 and 20 in Figures 3A and 3B (-1 and +1 in SEQ ID NO:6).
  • the potential leader sequence for the TRl 1SV2 protein shown in SEQ ID NO:6 is predicted to consist of amino acid residues -19 to -1 in SEQ ID NO:6, while the predicted mature TRl 1SV2 protein consists of amino acid residues 1 to 221 for the TR11SV2 protein shown in SEQ ID NO:6.
  • the TRl 1, TRl ISVl, and TRl 1SV2 receptor polypeptides encoded by the cDNAs of ATCC Deposit Numbers 209341, 209342, and 209343 comprise about 241 amino acids (but may be anywhere in the range of 224 to 251 amino acids), about 241 amino acids (but may be anywhere in the range of 231 to 251 amino acids), and about 240 amino acids (but may be anywhere in the range of 230 to 250 amino acids).
  • the predicted leader sequences of these proteins are about 25, 0, and 19 amino acids, but the actual leaders may be anywhere in the range of about 15 to about 35, about 20 to about 40, and about 9 to about 29 amino acids, respectively.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • nucleic acid contained in a clone that is a member of a library e.g., a genomic or cDNA library
  • a chromosome isolated or removed from a cell or a cell lysate e.g., a "chromosome spread", as in a karyotype
  • isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in Figures 1A and IB (SEQ ID NO:l); DNA molecules comprising the coding sequence for the mature TRl 1 receptor shown in Figures 1A and IB (SEQ ID NO:2; about the last 209 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRll receptor protein shown in Figure 1A (SEQ ID NO:2).
  • ORF open reading frame
  • SEQ ID NO:l DNA molecules comprising the coding sequence for the mature TRl 1 receptor shown in Figures 1A and IB
  • SEQ ID NO:2 DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRll receptor protein shown in Figure 1A (SEQ ID NO:2).
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in Figure 2A and 2B (SEQ ID NO:3); DNA molecules comprising the coding sequence for the mature TRl ISVl receptor shown in Figures 2 A and 2B (SEQ TD NO:4; about the last 241 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRl ISVl receptor protein shown in Figures 2A and 2B (SEQ ID NO:4).
  • ORF open reading frame
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in Figures 3A and 3B (SEQ ID NO:5); DNA molecules comprising the coding sequence for the mature TRl 1SV2 receptor shown in Figures 3A and 3B (SEQ ID NO:6; about the last 221 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRl 1SV2 receptor protein shown in Figures 3 A and 3B (SEQ ID NO:6).
  • ORF open reading frame
  • SEQ ID NO:5 DNA molecules comprising the coding sequence for the mature TRl 1SV2 receptor shown in Figures 3A and 3B
  • SEQ ID NO:6 DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRl 1SV2 receptor protein shown in Figures 3 A and 3B (SEQ ID NO:6).
  • the genetic code is well known in the
  • the invention provides isolated nucleic acid molecules encoding the TRl 1, TRl ISVl, and TRl 1SV2 polypeptides having the amino acid sequence encoded by the cDNA clones contained in the plasmids deposited as ATCC Deposit Nos. 209341, 209342, and 209343, respectively, on October 7, 1997.
  • these nucleic acid molecules will encode a mature polypeptide or the full-length polypeptide lacking the N-terminal methionine.
  • the invention further provides isolated nucleic acid molecules having the nucleotide sequences shown in Figures 1A and IB (SEQ ID NO:l), 2A and 2B (SEQ ID NO:3), and 3A and 3B (SEQ ID NO:5), the nucleotide sequences of the cDNAs contained in the above-described deposited clones; or nucleic acid molecules having a sequence complementary to one of the above sequences.
  • isolated molecules particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TRl 1, TRl ISVl, and TRl 1SV2 receptor genes of the present invention in human tissue, for instance, by Northern blot analysis.
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5 which have been determined from the following related cDNA clones: HHEAC71RA (SEQ ID NO:8) and HCFAZ22R (SEQ ID NO:9).
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequences shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • fragments 50-400 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs or as shown in Figures 1 A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively).
  • fragments which include 20 or more contiguous bases from the nucleotide sequences of the deposited cDNAs or the nucleotide sequence as shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively).
  • the present invention is also directed to an isolated fragment of a nucleic acid molecule, comprising a polynucleotide having a sequence shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively), or any sequence complementary to those shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively), wherein said fragment comprises at least 30 to 50 contiguous nucleotides from SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5, provided that said isolated nucleic acid molecule is not SEQ ID NO:8, SEQ ID NO:9 or any subfragment thereof.
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising the TRl 1 receptor protein of Figures 1A and IB (SEQ ID NO:2) extracellular domain (predicted to constitute amino acid residues from about 26 to about 162 in Figures 1A and IB (amino acid residues 1 to 137 in SEQ ID NO:2)); a polypeptide comprising the TRl ISVl receptor protein of Figures 2A and 2B (SEQ ID NO:4) extracellular domain (predicted to constitute amino acid residues from about 1 to about 162 in Figures 2A and 2B (amino acid residues 1 to 162 in SEQ ID NO:4)); a polypeptide comprising the TRl 1SV2 receptor protein of Figures 3A and 3B (SEQ ID NO:6) extracellular domain (predicted to constitute amino acid residues from about 20 to about 168 in Figures 3A and 3B (amino acid residues 1 to 149 in SEQ ID NO:6)); a poly
  • amino acid residues constituting the extracellular, transmembrane and intracellular domains have been predicted by computer analysis.
  • the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain.
  • nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the TRl 1 receptor proteins.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about Arg-2 to about Gly-11 in SEQ ED NO:2; a polypeptide comprising amino acid residues from about Thr-18 to about Arg-26 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Arg-34 to about Cys-42 in SEQ ID NO: 2; a polypeptide comprising amino acid residues from about Arg-31 to about Glu-39 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Gly-38 to about Asp-46 in SEQ ED NO:2; a polypeptide comprising amino acid residues from about Gly-74 to about Ser-82 in SEQ ID NO:2; a polypeptide comprising amino acid
  • nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the TRl ISVl receptor proteins.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about Ala-2 to about Ile-10 in SEQ ID NO:4; a polypeptide comprising amino acid residues from about Asn-11 to about Gly-19 in SEQ ID NO:4; a polypeptide comprising amino acid residues from about Thr-27 to about Ser-35 in SEQ ID NO:4; a polypeptide comprising amino acid residues from about Trp-38 to about Glu-46 in SEQ ID NO:4; a polypeptide comprising amino acid residues from about Gly-42 to about Ser-50 in SEQ ID NO:4; a polypeptide comprising amino acid residues from about Glu-31 to about Glu-46 in SEQ ED NO:4; a polypeptide comprising amino acid residues
  • nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the TRl 1SV2 receptor proteins.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about Gln-1 to about Cys-9 in SEQ ED NO:6; a polypeptide comprising amino acid residues from about Gly-5 to about Arg- 13 in SEQ ED NO:6; a polypeptide comprising amino acid residues from about Thr-18 to about Arg-26 in SEQ ID NO:6; a polypeptide comprising amino acid residues from about Thr-29 to about Pro-37 in SEQ ID NO:6; a polypeptide comprising amino acid residues from about Cys-48 to about Glu-56 in SEQ ID NO:6; a polypeptide comprising amino acid residues from about Val-87 to about Phe-95 in SEQ ED NO:6; a polypeptide comprising
  • nucleic acid molecules comprising, or alternatively consisting of a polypeptide sequence encoding amino acids 9 to 47, 49 to 86, or 90 to 128 of SEQ ED NO:2.
  • nucleic acid molecules of the invention comprise, or alternatively consist of, polynucleotide sequences encoding any combination of two, or all three of the above-recited TRl 1 cysteine-rich motifs. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridizes under stringent hybridization conditions to a portion of the polynucleotide of one of the nucleic acid molecules of the invention described above, for instance, the cDNA clones contained in ATCC Deposit Nos. 209341, 209342, and 209343, respectively.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • the reference polynucleotide e.g., the deposited cDNAs or the nucleotide sequences as shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively).
  • nucleic acid molecules of the present invention which encode
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides may include, but are not limited to those encoding the amino acid sequences of the mature polypeptides, by themselves; the coding sequences for the mature polypeptides and additional sequences, such as those encoding the potential leader or signal peptide sequences, such as pre-, or pro- or prepro- protein sequences; the coding sequences of the mature polypeptides, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to, introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequences encoding the polypeptides may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described by Gentz and colleagues (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)), for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson and coworkers (Cell 37:767 (1984)).
  • other such fusion proteins include the TRl 1 receptors fused to IgG- Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the TRl 1, TRl ISVl, and TRl 1SV2 receptors.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).
  • art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter
  • variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TRl 1, TRl ISVl, and TRl 1SV2 receptors or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 80%, 85% or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to: (a) a nucleotide sequence encoding the TRl 1 polypeptide having the complete amino acid sequence shown in Figures 1A and IB (amino acid residues -25 to 209 in SEQ ID NO:2); (b) a nucleotide sequence encoding the TRl ISVl polypeptide having the complete amino acid sequence shown in Figures 2A and 2B (amino acid residues 1 to 241 in SEQ ID NO:4); (c) a nucleotide sequence encoding the TRl 1SV2 polypeptide having the complete amino acid sequence shown in Figures 3A and 3B (amino acid residues -19 to 221 in SEQ ID NO:6); (d) a nucleotide encoding the complete amino sequence
  • nucleic acids of the invention comprise, or alternatively consist of, a polynucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding one, two, or all three of the cysteine-rich motifs described above (i.e., amino acids 9 to 47, 49 to 86, and/or 90 to 128 of SEQ ID NO:2).
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to one, two, or all three of the cysteine-rich motifs described above polynucleotides of the invention described above.
  • stringent conditions as used herein is described infra.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • a further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of a TRl 1 , TRl ISVl and/or TRl 1SV2 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably not more than 30 conservative amino acid substitutions, and still even more preferably not more than 20 conservative amino acid substitutions.
  • a polynucleotide which encodes the amino acid sequence of a TRl 1, TRl ISVl or TRl 1SV2 polypeptide to have an amino acid sequence which contains not more than 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TRl 1, TRl ISVl or TRl 1SV2 polypeptides or peptides by recombinant techniques.
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the TR 11 , TR 11 S V 1 or TRl 1 S V2 receptors.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in Figures 1 A and IB, 2A and 2B, and/or 3 A and 3B, or to the nucleotides sequence of the deposited cDNA clones can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman to find the best segment of homology between two sequences (Advances in Applied Mathematics 2:482-489 (1981)).
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
  • the present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shown in Figures 1A and IB, 2A and 2B, and 3 A and 3B (SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively) or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having TRl 1, TRl ISVl or
  • TRl 1SV2 receptor activity is also directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides set forth herein as n'-m 1 , n 2 -m 2 , n 3 -m 3 , n 4 -m 4 , n 5 -m 5 , n 6 -m 6 , and/or n 7 -m 7 . Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TRl 1, TRl ISVl or TRl 1SV2 receptor activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having TRl 1, TRl ISVl or TRl 1SV2 receptor activity include, inter alia, (1) isolating a TRl 1, TRl ISVl or TRl 1SV2 receptor gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of a TRl 1, TRl ISVl or TRl 1SV2 receptor gene, as described by Verma and colleagues (Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988)); and (3) Northern Blot analysis for detecting TRl 1, TRl ISVl or TRl 1SV2 receptor mRNA expression in specific tissues.
  • a polypeptide having TRll, TRl ISVl, and TR11SV2 receptor activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TRl l, TRl lSVl, and TRl 1SV2 receptors of the present invention (either the full-length protein, the splice variants, or, preferably, the mature protein), as measured in a particular biological assay.
  • TRl 1, TRl ISVl, and TRl 1SV2 receptor activities can be measured by determining the ability of a TR 11 , TR 11 S V 1 , or TR 11 S V2 polypeptide- Fc fusion protein to inhibit lymphocyte (e.g., T cell) proliferation, differentiation or activation and/or to extend T cell survival.
  • TRl 1, TRl ISVl, and TRl 1SV2 receptor activities may also be measured by determining the ability of a polypeptide, such as cognate ligand which is free or expressed on a cell surface, to confer proliferatory activity, and/or increase cell survival, in intact cells expressing one or more of the receptors.
  • a polypeptide such as cognate ligand which is free or expressed on a cell surface
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences of the deposited cDNAs or the nucleic acid sequence shown in Figures 1A and IB, 2A and 2B, and 3A and 3B will encode polypeptides "having TRll, TRllSVl or TRl 1SV2 receptor activity.”
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • Vectors and Host Cells The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of TRl 1, TRl ISVl, and TRl 1SV2 polypeptides or fragments thereof by recombinant or synthetic techniques.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • suitable heterologous hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pHE4 (ATCC Accession Number 209645, deposited February 25, 1998), pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, CA).
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • TRl l, TRl lSVl, and/or TR11SV2 polypeptides, and preferably the secreted form can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • the yeast Pichia pastoris is used to express TRl 1
  • Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
  • a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 .
  • alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al, Yeast 5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76 (1987).
  • a heterologous coding sequence such as, for example, a TRl l, TRllSVl, and/or TRl 1SV2 polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
  • the plasmid vector pPIC9K is used to express DNA encoding a
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptide of the invention as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998.
  • This expression vector allows expression and secretion of a TRl 1, TRl ISVl, and/or TRl 1SV2 protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
  • PHO Pichia pastoris alkaline phosphatase
  • yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in- frame AUG as required.
  • high-level expression of a heterologous coding sequence such as, for example, a TRl 1, TRl ISVl, and or TRl 1SV2 polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
  • the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides may be glycosylated or may be non- glycosylated.
  • TRl l, TRllSVl, and or TR11SV2 polypeptides may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
  • the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.
  • N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., TRl 1,
  • TRl lSVl and/or TRl 1SV2 coding sequence and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TRll, TRl ISVl and/or TRl 1SV2 polynucleotides of the invention, and which activates, alters, and or amplifies endogenous TRll, TRllSVl and or TR11SV2 polynucleotides.
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous control regions e.g., promoter and/or enhancer
  • TRl 1, TRl ISVl and/or TRl 1SV2 polynucleotide sequences via homologous recombination
  • heterologous control regions e.g., promoter and/or enhancer
  • TRl 1, TRl ISVl and/or TRl 1SV2 polynucleotide sequences via homologous recombination
  • heterologous control regions e.g., promoter and/or enhancer
  • TRl 1, TRl ISVl and/or TRl 1SV2 polynucleotide sequences via homologous recombination
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as, human hIL-5 receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson et al, J. Biol. Chem., 270(16):9459-9471 (1995).
  • TRl 1, TRl ISVl and/or TRl 1SV2 polypeptides of the invention comprise fusion proteins as described above wherein the
  • TRl 1, TRl ISVl and or TRl 1SV2 polypeptides are those described as n'-m 1 , n 2 -m 2 , n 3 -m ⁇ n 4 -m 4 , n 5 -m 5 , n 6 -m 6 , n 7 -m 7 herein.
  • the invention is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein.
  • polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310: 105-111).
  • a peptide corresponding to a fragment of the TRl 1, TRl ISVl and/or TRl 1SV2 polypeptides of the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TRl 1, TRl ISVl and/or TRl 1SV2 polynucleotide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4- diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al, Nucl. Acids Res. 7A4331 (1986); and Zoller et al, Nucl. Acids Res.
  • TRl 1 , TRl ISVl and/or TRl 1SV2 polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the 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, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
  • the polyethylene glycol may have a branched structure.
  • Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 78:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. 70:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art, e.g., EP 0401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • pegylation of the proteins of the invention may be accomplished by any number of means.
  • polyethylene glycol may be attached to the protein either directly or by an intervening linker.
  • Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol. 68:1- 18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • ClSO 2 CH 2 CF 3 tresylchloride
  • polyethylene glycol is directly attached to amine groups of the protein.
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
  • U.S. Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with l,l'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • the number of polyethylene glycol moieties attached to each protein of the invention may also vary.
  • the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
  • the average degree of substitution within ranges such as 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 moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249- 304 (1992).]
  • TRl 1, TRl ISVl and TRl 1SV2 receptors can be recovered and purified from by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • polypeptides of the invention can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
  • transgene i.e., polynucleotides of the invention
  • transgene i.e., polynucleotides of the invention
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830- 834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl.
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
  • Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TRl l, TRl lSVl and or TR11SV2 polypeptides, studying conditions and/or disorders associated with aberrant TRl 1, TRl ISVl and/or TRl 1SV2 expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
  • the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., 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, for example, Anderson et al. U.S. Patent No. 5,399,349; and Mulligan & Wilson, U.S. Patent No. 5,460,959, each of which is incorporated by reference herein in its entirety).
  • the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • TRll, TRllSVl, and TR11SV2 Polypeptides and Fragments The TRl 1 polypeptides of the invention may be in monomers or multimers
  • the present invention relates to monomers and multimers of the TRl 1, TRl ISVl and/or TRl 1SV2 polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
  • the polypeptides of the invention are monomers, dimers, trimers or tetramers.
  • the multimers of the invention are at least dimers, at least trimers, or at least tetramers. Multimers encompassed by the invention may be homomers or heteromers.
  • homomer refers to a multimer containing only TRl l, TRllSVl and or TRl 1SV2 polypeptides of the invention (including fragments, variants, and fusion proteins, as described herein, of TRl 1, TRl ISVl and/or TRl 1SV2).
  • a TRl 1 homomer will contain only TRl 1 polypeptides of the invention (including fragments, variants, and fusion proteins, as described herein, of TRl 1), whereas a TRl ISVl homomer will contain only TRl ISVl polypeptides of the invention (including fragments, variants, and fusion proteins, as described herein, of TRl ISVl), and a TRl 1SV2 homomer will contain only TRl 1SV2 polypeptides of the invention (including fragments, variants, and fusion proteins, as described herein, of TRl 1SV2).
  • homomers may contain TRl 1 , TRl ISVl and or TRl 1SV2 polypeptides having identical or different amino acid sequences.
  • a homomer of the invention is a multimer containing only TRl 1 , TRllSVl and/or TR11SV2 polypeptides having an identical amino acid sequence.
  • a homomer of the invention is a multimer containing TRl l, TRllSVl and/or TRl IS V2 polypeptides having different amino acid sequences.
  • the multimer of the invention is a homodimer (e.g., containing TRll, TRllSVl and/or TR11SV2 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing TRl 1, TRl ISVl and or TRl 1SV2 polypeptides having identical or different amino acid sequences).
  • the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • heteromer refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the TRll, TRl ISVl and/or TRl 1SV2 polypeptides of the invention.
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
  • the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers of the invention such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention
  • multimers of the invention are formed by covalent associations with and/or between the TRl 1, TRl ISVl and/or TRl 1SV2 polypeptides of the invention.
  • covalent associations may involve one or more amino acid residues contained in the TRl 1, TRl ISVl and or TRl 1SV2 polypeptide sequences ( e.g., those recited in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or contained in the respective TRl 1, TRl ISVl and TRl 1SV2 polypeptides encoded by the respective clones HHEAC71, HCFAZ22, and HT5EA78).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TRl 1, TRl ISVl or TRl 1SV2 fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
  • covalent associations are between the heterologous sequence contained in a TRl 1-Fc, TRl lSVl-Fc or TRl lSV2-Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
  • two or more TRl 1, TRl ISVl, TRl 1SV2 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers).
  • synthetic linkers e.g., peptide, carbohydrate or soluble polymer linkers.
  • proteins comprising multiple TRl 1, TRl ISVl, TRl 1SV2 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • TRl 1, TRl ISVl, TRl 1SV2 polypeptides of the invention involves use of TRll, TRllSVl, TR11SV2 polypeptides fused to a leucine zipper or isoleucine zipper polypeptide sequence.
  • Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins.
  • leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric TRl 1, TRl ISVl, TRl 1SV2 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble TRl 1, TRl ISVl, TRl 1SV2 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TRl l, TRllSVl, TR11SV2 is recovered from the culture supernatant using techniques known in the art.
  • trimeric TRll, TRllSVl, TR11SV2 may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers (e.g., isoleucine zippers).
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
  • proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in Flag®-TR11, TRl ISVl , TRl 1SV2 or Flag®-TR11, TRl ISVl, TRl 1SV2 fusion proteins of the invention.
  • associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag®-TR11, TRllSVl, TR11SV2 or Flag®-TR11, TRl lSVl, TR11SV2 fusion proteins of the invention and anti-Flag® antibody.
  • the multimers of the invention may be generated using chemical techniques known in the art.
  • polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • linker molecules and linker molecule length optimization techniques known in the art
  • multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers of the invention may be generated using genetic engineering techniques known in the art.
  • polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • TRl 1 polynucleotide fragments include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501- 550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950 or 951 to the end of SEQ ID NO:l or the cDNA contained in the deposited clone.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • TRl ISVl polynucleotide fragments include, for example, fragments having a sequence selected from the group from about nucleotide number 1- 50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451- 500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1007 or 951 to the end of SEQ ID NO:3 or the cDNA contained in the deposited clone. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • TRl 1SV2 polynucleotide fragments include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501- 550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051 to the end of SEQ ID NO:5 or the cDNA contained in the deposited clone. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • polypeptide fragment refers to a amino acid sequence contained in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or encoded by the cDNA contained in the deposited clones.
  • Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments having a sequence selected from the group from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221 to the end of the coding region of SEQ ID NO:2; 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141- 160, 161-180, 181-200, 201-220, 221 to the end of the coding region of SEQ ID NO:4; or 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220, and 221 to the
  • polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • Polynucleotides encoding these polypeptide fragments of the invention are also encompassed by the invention.
  • polypeptides of the invention comprise, or alternatively consist of, amino acid residues Met-1 to Pro-62, Ala-2 to Pro-62, Met-1 to Ser-50, Ala-2 to Ser-50, Ser-50 to Pro-62, Met-1 to Trp-196, Met-1 to Gln-197, Met-1 to Leu-198, Met-1 to Arg-199, Met-1 to Lys-200, Met-1 to Thr-201, Met-1 to Gln-202, Met-1 to Leu-203, Met-1 to Leu-204, Met-1 to Leu-205, Ala-2 to Trp-196, Ala-2 to Gln-197, Ala-2 to Leu-198, Ala-2 to Arg-199, Ala-2 to Lys-200, Ala-2 to Thr-201, Ala-2 to Gln-202, Ala-2 to Leu-203, Ala-2 to Leu-204, Ala-2 to Leu-205, Ser-50 to Trp-196, Ser-50 to Gln-197, Ser-50 to Leu-197, Ser
  • polynucleotides encoding these specific polypeptide fragments are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRll, TRllSVl, and/or TRl IS V2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:l SEQ ID NO:3 and or SEQ ID NO:5
  • SEQ ID NO:5 SEQ ID NO:5
  • a 1 is any integer between 1 to 969 of SEQ ID NO:l
  • b 1 is an integer of 15 to 983, where both a 1 and b 1 correspond to the positions of nucleotide residues shown in SEQ ID NO:l, and where the b 1 is greater than or equal to a 1 + 14.
  • a 2 is any integer between 1 to 993 of SEQ ID NO:3, b 2 is an integer of 15 to 1007, where both a 2 and b 2 correspond to the positions of nucleotide residues shown in SEQ ID NO:3, and where the b 2 is greater than or equal to a 2 + 14.
  • a 3 is any integer between 1 to 1060 of SEQ ID NO:5
  • b 3 is an integer of 15 to 1074, where both a 3 and b 3 correspond to the positions of nucleotide residues shown in SEQ ID NO:5, and where the b 3 is greater than or equal to a 3 + 14.
  • polynucleotides of the invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.
  • polynucleotides of the invention comprise at least 15 contiguous nucleotides of TRl 1, TRl ISVl, or TRl 1SV2 coding sequence, but do not comprise all or a portion of any TRl 1, TRl ISVl, or TRl 1SV2 intron.
  • the nucleic acid comprising TRl 1, TRl ISVl, or TRl 1SV2 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TRl l, TRl lSVl, or TR11SV2 gene in the genome).
  • the polynucleotides of the invention are less than 100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.
  • polynucleotides of the invention comprise 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 TRll, TRllSVl or TR11SV2 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide sequences set forth in Figures 1A and IB (SEQ ID NO:l), Figures 2A and 2B (SEQ ID NO:3), and Figures 3A and 3B (SEQ ID NO:5), respectively.
  • polynucleotides of the invention comprise 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 TRl 1 , TRl ISVl and or TRl 1SV2 coding sequence, but do not comprise all or a portion of any TRl 1, TRl ISVl and or TRl 1SV2 intron.
  • the nucleic acid comprising TRl 1, TRl ISVl and/or TRl 1SV2 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TRl 1, TRl ISVl and/or TRl 1SV2 gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • the invention further provides isolated TRl 1, TRl ISVl, and TRl 1SV2 polypeptides having the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequences in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively) or a peptide or polypeptide comprising a portion of the above polypeptides.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides protein engineering may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins, including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six TRl 1, TRl ISVl or TRl 1SV2 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TRl 1 amino acid sequence shown in Figures 1A and IB (SEQ ID NO:2), up to the leucine residue at position number 229 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n'-234 of Figures 1A and IB (SEQ ED NO:2), where n 1 is an integer in the range of 2 to 229, and 230 is the position of the first residue from the N-terminus of the complete TRl 1 polypeptide believed to be required for at least immunogenic activity of the TRl 1 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of A-2 to V-234; Q-3 to V-234; H-4 to V-234; G-5 to V-234; A-6 to V-234; M-7 to V-234; G-8 to V-234; A-9 to V-234; F-10 to V-234; R-11 to V-234; A-12 to V-234; L-13 to V-234; C-14 to V-234; G-15 to V-234; L-16 to V-234; A-17 to V-234; L-18 to V-234; L-19 to V-234; C-20 to V-234; A-21 to V-234; L-22 to V-234; S-23 to V-234; L-24 to V-234; G-25 to V-234; Q-26 to V-234; R-27 to V-234; P-28 to V-234; T-29 to V-234; G-30 to V-2
  • V-234 P-32 to V-234; G-33 to V-234; C-34 to V-234; G-35 to V-234; P-36 to V-234;
  • V-234 T-43 to V-234; G-44 to V-234; T-45 to V-234; D-46 to V-234; A-47 to V-234;
  • V-234 T-54 to V-234; T-55 to V-234; R-56 to V-234; C-57 to V-234; C-58 to V-234;
  • V-234 E-65 to V-234; C-66 to V-234; C-67 to V-234; S-68 to V-234; E-69 to V-234;
  • V-234 C-81 to V-234; G-82 to V-234; D-83 to V-234; P-84 to V-234; C-85 to V-234;
  • V-234 H-92 to V-234; P-93 to V-234; C-94 to V-234; P-95 to V-234; P-96 to V-234;
  • V-142 to V-234 F-143 to V-234; P-144 to V-234; G-145 to V-234; N-146 to V-234;
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRllSVl, and or TR11SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TRl 1 shown in Figures 1 A and IB (SEQ ID NO:2), up to the alanine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 1 of Figures 1A and IB (SEQ ED NO:2), where m 1 is an integer in the range of 6 to 234, and 6 is the position of the first residue from the C-terminus of the complete TRl 1 polypeptide believed to be required for at least immunogenic activity of the TRll protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to W-233; M-1 to L-232; M-1 to D-231; M-1 to G-230; M-1 to L-229; M-1 to R-228; M-1 to G-227; M-1 to K-226; M-1 to E-225; M-1 to E-224; M-1 to A-223; M-1 to S-222; M-1 to R-221; M-1 to E-220; M-1 to G-219; M-1 to R-218; M-1 to E-217; M-1 to E-216; M-1 to E-215; M-1 to P-214; M-1 to F-213; M-1 to Q-212; M-1 to C-211; M-1 to S-210; M-1 to R-209; M-1 to A-208; M-1 to D-207; M-1 to E-206; M-1 to T-205; M-1 to S
  • M-1 to F-106 M-1 to K-105; M-1 to G-104; M-1 to Q-103; M-1 to S-102; M-1 to Q-101; M-1 to V-100; M-1 to G-99; M-1 to Q-98; M-1 to G-97; M-1 to P-96; M-1 to P-95; M-1 to C-94; M-1 to P-93; M-1 to H-92; M-1 to H-91; M-1 to R-90; M-1 to C-89; M-1 to T-88; M-1 to T-87; M-1 to C-86; M-1 to C-85; M-1 to P-84; M-1 to D-83; M-1 to G-82; M-1 to C-81; M-1 to H-80; M-1 to F-79; M-1 to E-78; M-1 to P-77; M-1 to Q-76; M-1 to V-75; M-1 to C-74; M-1 to M-73; M-1 to C-72; M-1 to D-71
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRll, TRllSVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TRl 1 polypeptide, which may be described generally as having residues n'-m 1 of Figures 1A and IB (SEQ ID NO:2), where n 1 and m 1 are integers as described above.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TRllSVl amino acid sequence shown in SEQ ID NO:4, up to the leucine residue at position number 236 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 2 -241 of Figures 2A and 2B (SEQ ID NO:4), where n 2 is an integer in the range of 2 to 236, and 237 is the position of the first residue from the N-terminus of the complete TRl ISVl polypeptide believed to be required for at least immunogenic activity of the TRl ISVl protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of A-2 to V-241 ; P-3 to V-241; G-4 to V-241; E-5 to V-241; R-6 to V-241; D-7 to V-241; S-8 to V-241; W-9 to V-241; 1-10 to V-241; N-l l to V-241; P-12 to V-241; G-13 to V-241; P-14 to V-241; D-15 to V-241; S-16 to V-241; Q-17 to V-241; P-18 to V-241; G-19 to V-241; A-20 to V-241; L-21 to V-241; C-22 to V-241; S-23 to V-241; L-24 to V-241; E-25 to V-241; P-26 to V-241; T-27 to V-241; V-28 to V-241; G-29 to V-241; G
  • G-118 to V-241 T-119 to V-241; F-120 to V-241; S-121 to V-241; G-122 to V-241; G-123 to V-241 H-124 to V-241; E-125 to V-241; G-126 to V-241; H-127 to V-241; C-128 to V-241 K-129 to V-241; P-130 to V-241; W-131 to V-241; T-132 to V-241; D-133 to V-241 C-134 to V-241; T-135 to V-241; Q-136 to V-241; F-137 to V-241; G-138 to V-241 F-139 to V-241; L-140 to V-241; T-141 to V-241; V-142 to V-241; F-143 to V-241 p-144 to V-241; G-145 to V-241; N-146 to V-241; K-147 to V-241; T-148 to V-241 H-149 to V-241; N-150 to
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TRl lSVl shown in SEQ ID NO:4, up to the arginine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 2 of Figures 2A and 2B (SEQ ID NO:4), where m 2 is an integer in the range of 6 to 241, and 6 is the position of the first residue from the C-terminus of the complete TRl ISVl polypeptide believed to be required for at least immunogenic activity of the TRl ISVl protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to W-240; M-1 to L-239; M-1 to D-238; M-1 to G-237; M-1 to L-236; M-1 to R-235; M-1 to G-234; M-1 to K-233; M-1 to E-232; M-1 to E-231; M-1 to A-230; M-1 to S-229; M-1 to R-228; M-1 to E-227; M-1 to G-226; M-1 to R-225; M-1 to E-224; M-1 to E-223; M-1 to E-222; M-1 to P-221; M-1 to F-220; M-1 to Q-219; M-1 to C-218; M-1 to S-217; M-1 to R-216; M-1 to A-215; M-1 to D-214; M-1 to E-213; M-1 to T-212; M-1 to S
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TRl ISVl polypeptide, which may be described generally as having residues n 2 -m 2 of Figures 2A and 2B (SEQ ID NO:4), where n 2 and m 2 are integers as described above.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TRl 1SV2 amino acid sequence shown in Figures 3A and 3B (SEQ ID NO:6), up to the leucine residue at position number 235 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 3 -240 of Figures 3A and 3B (SEQ ID NO:6), where n 3 is an integer in the range of 2 to 235, and 236 is the position of the first residue from the N-terminus of the complete TRl 1SV2 polypeptide believed to be required for at least immunogenic activity of the TRl 1SV2 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of G-2 to V-240; A-3 to V-240; F-4 to V-240; R-5 to V-240; A-6 to V-240; L-7 to V-240; C-8 to V-240; G-9 to V-240; L-10 to V-240; A-ll to V-240; L-12 to V-240; L-13 to V-240; C-14 to V-240; A-15 to V-240; L-16 to V-240; S-17 to V-240; L-18 to V-240; G-19 to V-240; Q-20 to V-240; R-21 to V-240; P-22 to V-240; T-23 to V-240; G-24 to V-240; G-25 to V-240; P-26 to V-240; G-27 to V-240; C-28 to V-240; G-29 to V-240; P-30 to V
  • V-240 E-71 to V-240; C-72 to V-240; C-73 to V-240; S-74 to V-240; E-75 to V-240;
  • V-240 C-87 to V-240; G-88 to V-240; D-89 to V-240; P-90 to V-240; C-91 to V-240;
  • V-240 S-123 to V-240 G-124 to V-240; T-125 to V-240; F-126 to V-240; S-127 to
  • V-240 A-178 to V-240 V-179 to V-240; A-180 to V-240; A-181 to V-240; C-182 to
  • V-240 V-183 to V-240 L-184 to V-240; L-185 to V-240; L-186 to V-240; T-187 to
  • V-240 G-233 to V-240 R-234 to V-240; and L-235 to V-240 of the TRl 1SV2 amino acid sequence shown in Figures 3A and 3B (which is identical to the sequence shown as SEQ ID NO:6, with the exception that the amino acid residues in Figures 3A and 3B are numbered consecutively from 1 through 240 from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:6 are numbered consecutively from -19 through 221 to reflect the position of the predicted signal peptide).
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TRl 1SV2 shown in Figures 3A and 3B (SEQ ID NO:6), up to the alanine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 3 of Figures 3A and 3B (SEQ ED NO:6), where m 3 is an integer in the range of 6 to 240, and 6 is the position of the first residue from the C-terminus of the complete TRl 1SV2 polypeptide believed to be required for at least immunogenic activity of the TRl 1SV2 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to W-239; M-1 to L-238; M-1 to D-237; M-1 to G-236; M-1 to L-235; M-1 to R-234; M-1 to G-233; M-1 to
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TRl 1SV2 polypeptide, which may be described generally as having residues n 3 -m 3 of Figures 3 A and 3B (SEQ ID NO:6), where n 3 and m 3 are integers as described above.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TRl 1 amino acid sequence shown in Figures 1 A and IB (SEQ ID NO:2), up to the glycine residue at position number 156 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 4 -162 of Figures 1A and IB (SEQ ID NO:2), where n 4 is an integer in the range of 25 to 156, and 157 is the position of the first residue from the N-terminus of the predicted extracellular domain of the TRl 1 polypeptide believed to be required for at least immunogenic activity of the predicted extracellular domain of the TRl 1 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of G-25 to P-162; Q-26 to P-162; R-27 to P-162; P-28 to P-162; T-29 to P-162; G-30 to P-162; G-31 to P-162; P-32 to P-162; G-33 to P-162; C-34 to P-162; G-35 to P-162; P-36 to P-162; G-37 to P-162; R-38 to P-162; L-39 to P-162; L-40 to P-162; L-41 to P-162; G-42 to P-162; T-43 to P-162; G-44 to P-162; T-45 to P-162; D-46 to P-162; A-47 to P-162; R-48 to P-162; C-49 to P-162; C-50 to P-162; R-
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TRl 1 shown in Figures 1 A and IB (SEQ ID NO:2), up to the glycine residue at position number 31, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 25-m 4 of Figures 1A and IB (SEQ ID NO:2), where m 4 is an integer in the range of 31 to 162, and 30 is the position of the first residue from the C-terminus of the predicted extracellular domain of the TRl 1 polypeptide believed to be required for at least immunogenic activity of the TRl 1 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues G-25 to P-162; G-25 to E-161; G-25 to A-160; G-25 to P-159; G-25 to P-158; G-25 to S-157; G-25 to G-156; G-25 to P-155; G-25 to V-154; G-25 to C-153; G-25 to V-152; G-25 to A-151; G-25 to N-150; G-25 to H-149; G-25 to T-148; G-25 to K-147; G-25 to N-146; G-25 to G-145; G-25 to P-144; G-25 to F-143; G-25 to V-142; G-25 to T-141; G-25 to L-140; G-25 to F-139; G-25 to G-138; G-25 to F-137; G-25 to Q-136; G-25 to T-135; G-25 to C-134; G-25 to D
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TRl 1 polypeptide, which may be described generally as having residues n 4 -m 4 of Figures 1A and IB (SEQ ED NO:2), where n 4 and m 4 are integers as described above.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TRl ISVl amino acid sequence shown in Figures 2A and 2B (SEQ ED NO:4), up to the glycine residue at position number 156 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 5 -162 of Figures 2A and 2B (SEQ ID NO:4), where n 5 is an integer in the range of 1 to 156, and 157 is the position of the first residue from the N-terminus of the predicted extracellular domain of the TRl ISVl polypeptide believed to be required for at least immunogenic activity of the predicted extracellular domain of the TRl ISVl protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of M-1 to P-162; A-2 to P-162; P-3 to P-162; G-4 to P-162; E-5 to P-162; R-6 to P-162; D-7 to P-162; S-8 to P-162; W-9 to P-162; 1-10 to P-162; N-l l to P-162; P-12 to P-162; G-13 to P-162; P-14 to P-162; D-15 to P-162; S-16 to P-162; Q-17 to P-162; P-18 to P-162; G-19 to P-162; A-20 to P-162; L-21 to P-162; C-22 to P-162; S-23 to P-162; L-24 to P-162; E-25 to P-162; P-26 to P-162; T-27 to P-162; V-28 to P-162; G-29
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TRl ISVl shown in Figures 2A and 2B (SEQ ID NO:4), up to the arginine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 5 of Figures 2A and 2B (SEQ ED NO:4), where m 5 is an integer in the range of 6 to 162, and 6 is the position of the first residue from the C-terminus of the predicted extracellular domain of the TRl ISVl polypeptide believed to be required for at least immunogenic activity of the TRl ISVl protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to P-162; M-1 to E-161; M-1 to A-160; M-1 to P-159; M-1 to P-158; M-1 to S-157; M-1 to G-156; M-1 to
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TRl ISVl polypeptide, which may be described generally as having residues n 5 -m 5 of Figures 2A and 2B (SEQ ID NO:4), where n 5 and m 5 are integers as described above.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TRl 1SV2 amino acid sequence shown in Figures 3 A and 3B (SEQ ID NO:6), up to the glycine residue at position number 162 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 5 -168 of Figures 3A and 3B (SEQ ID NO:6), where n 6 is an integer in the range of 20 to 162, and 163 is the position of the first residue from the N-terminus of the predicted extracellular domain of the TRl 1SV2 polypeptide believed to be required for at least immunogenic activity of the predicted extracellular domain of the TRl 1SV2 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of Q-20 to P-168; R-21 to P-168; P-22 to P-168; T-23 to P-168; G-24 to P-168; G-25 to P-168; P-26 to P-168; G-27 to P-168; C-28 to P-168; G-29 to P-168; P-30 to P-168; G-31 to P-168; R-32 to P-168; L-33 to P-168; L-34 to P-168; L-35 to P-168; G-36 to P-168; T-37 to P-168; G-38 to P-168; T-39 to P-168; D-40 to P-168; A-41 to P-168; R-42 to P-168; C-43 to P-168; C-44 to P-168; R-45 to P-168; V-46 to P
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRl ISVl, and/or TR11SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TR11SV2 shown in Figures 3A and 3B (SEQ ID NO:6), up to the proline residue at position number 26, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 20-m 6 of Figures 3A and 3B (SEQ ID NO:6), where m 6 is an integer in the range of 26 to 168, and 26 is the position of the first residue from the C-terminus of the predicted extracellular domain of the TRl 1SV2 polypeptide believed to be required for at least immunogenic activity of the TRl 1SV2 protein.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues Q-20 to P-168; Q-20 to E-167; Q-20 to A-166; Q-20 to P-165; Q-20 to P-164; Q-20 to S-163; Q-20 to G-162; Q-20 to P-161; Q-20 to V-160; Q-20 to C-159; Q-20 to V-158; Q-20 to A-157; Q-20 to N-156; Q-20 to H-155; Q-20 to T-154; Q-20 to K-153; Q-20 to N-152; Q-20 to G-151; Q-20 to P-150; Q-20 to F-149; Q-20 to V-148; Q-20 to T-147; Q-20 to L-146; Q-20 to F-145; Q-20 to G-144; Q-20 to F-143; Q-20 to Q-142; Q-20 to T-141; Q-20 to C-140; Q-20 to D
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRl 1, TRllSVl, and or TR11SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TRl 1SV2 polypeptide, which may be described generally as having residues n 6 -m 6 of Figures 3A and 3B (SEQ ID NO:6), where n 6 and m 6 are integers as described above.
  • the TRl 1 polynucleotide of the invention has the polynucleotide sequence shown in SEQ ID NO:28.
  • the corresponding TRl 1 polypeptide has the polypeptide sequence shown in SEQ ID NO:28.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TRl 1 amino acid sequence shown as SEQ ID NO:28, up to the leucine residue at position number 236 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 7 -241 of SEQ ID NO:28, where n 7 is an integer in the range of 2 to 236, and 237 is the position of the first residue from the N-terminus of the complete TRl 1 polypeptide believed to be required for at least immunogenic activity of the TRl 1 polypeptide.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues of A-2 to V-241 ; Q-3 to V-241 ; H-4 to V-241; G-5 to V-241; A-6 to V-241; M-7 to V-241; G-8 to V-241; A-9 to V-241; F-10 to V-241; R-ll to V-241; A-12 to V-241; L-13 to V-241; C-14 to V-241; G-15 to V-241; L-16 to V-241; A-17 to V-241; L-18 to V-241; L-19 to V-241; C-20 to V-241; A-21 to V-241; L-22 to V-241; S-23 to V-241; L-24 to V-241; G-25 to V-241; Q-26 to V-241; R-27 to V-241; P-28 to V-241; T-29 to V-241; G-30
  • V-142 to V-241 F-143 to V-241; P-144 to V-241; G-145 to V-241; N-146 to V-241;
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
  • TRl 1SV2 polypeptides described above and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • TRl 1 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six TRl 1 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TRl 1 polypeptide shown as SEQ ID NO:28, up to the alanine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 7 of SEQ ID NO:28, where m 7 is an integer in the range of 6 to 240, and 6 is the position of the first residue from the C-terminus of the complete TRl 1 polypeptide believed to be required for at least immunogenic activity of the TRl 1 polypeptide.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues M-1 to W-240; M-1 to L-239; M-1 to D-238; M-1 to G-237; M-1 to L-236; M-1 to R-235; M-1 to G-234; M-1 to K-233; M-1 to E-232; M-1 to E-231; M-1 to A-230; M-1 to S-229; M-1 to R-228; M-1 to E-227; M-1 to G-226; M-1 to R-225; M-1 to E-224; M-1 to E-223; M-1 to E-222; M-1 to P-221; M-1 to F-220; M-1 to Q-219; M-1 to C-218; M-1 to S-217; M-1 to
  • polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TRll, TRllSVl, and/or TRl 1SV2 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TRll polypeptide, which may be described generally as having residues n 7 -m 7 of SEQ ID NO:28, where n and m are integers as described above.
  • the polypeptides of this invention may be membrane bound or may be in a soluble circulating form. Soluble peptides are defined by amino acid sequence wherein the sequence comprises the polypeptide sequence lacking the transmembrane domain.
  • the polypeptides of the present invention may exist as a membrane bound receptor having a transmembrane region and an intra- and extracellular region or they may exist in soluble form wherein the transmembrane domain is lacking.
  • TRl 1, TRl ISVl, and TRl 1SV2 receptors is the TRl 1, TRllSVl, and TR11SV2 receptors shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively) which contain transmembrane, intracellular and extracellular domains.
  • these forms of the TRl 1, TRl ISVl, and TRl 1SV2 receptors appear to be localized in the cytoplasmic membrane of cells which express these proteins.
  • the invention further includes variations of the TRl 1, TRl ISVl, and TRl 1SV2 receptors which show substantial TRl 1, TRl ISVl or TRl 1SV2 receptor activities or which include regions of TRll, TRl lSVl, and TRl 1SV2 proteins such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • the fragments, derivatives or analogs of the polypeptides of Figures 1A and IB, 2A and 2B, and 3A and 3B may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence
  • TRl 1, TRl ISVl, and TRl 1SV2 receptors of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table I).
  • Embodiments of the invention are directed to polypeptides which comprise the amino acid sequence of a TRll, TRllSVl, and or TRl 1SV2 polypeptide described herein, but having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions, when compared with the TRl 1, TRl ISVl, and/or TRl 1SV2 polynucleotide sequence described herein.
  • a peptide or polypeptide in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of a TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • Amino acids in the TRl 1, TRl ISVl and TRl 1SV2 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
  • TRl 1 TRl ISVl and/or TRl 1SV2 polypeptides
  • protein engineering may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl. Acids Res. 13:4331 (1986); and Zoller et al, Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et ai, Philos. Trans. R. Soc. London SerA 317:415 (1986)).
  • art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter e
  • the invention also encompasses TRl l, TRllSVl and/or TRl 1SV2 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TRl 1 , TRl ISVl and/or TRl 1SV2 polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges;
  • N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • amino acid residues of the polypeptides of the invention may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.
  • DNA shuffling may be employed to modulate the activities of TRll, TRllSVl and/or TR11SV2 thereby effectively generating agonists and antagonists of TRl 1 , TRl ISVl and/or TRl 1SV2.
  • DNA shuffling may be employed to modulate the activities of TRll, TRllSVl and/or TR11SV2 thereby effectively generating agonists and antagonists of TRl 1 , TRl ISVl and/or TRl 1SV2.
  • U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten P. A., et al, Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al, J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.
  • alteration of TRl 1 , TRl ISVl and/or TRl 1SV2 polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments into a desired TRl 1, TRl ISVl and/or TRl 1SV2 molecule by homologous, or site-specific, recombination.
  • TRl 1, TRl ISVl and/or TRl 1SV2 polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of TRl l, TRllSVl and/or TRl 1SV2 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the heterologous molecules are, for example, TNF-alpha, lymphotoxin- alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6): 1185-1190), endokine-alpha (International Publication No.
  • WO 98/07880 Neutrokine-alpha (International Publication No. WO 98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694),TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No.
  • WO 98/54202 International Publication No. WO 98/06842
  • TR12 and TNF-RI
  • TRAMP/DR3/APO- 3/WSL/LARD International Publication No. WO 98/06842
  • TRAMP/DR3/APO- 3/WSL/LARD TRAIL-R1/DR4/APO-2
  • TRAEL-R2/DR5 DcRl/TRAEL- R3/TRID/LIT
  • DcR2/TRAIL-R4 CAD
  • TRAIL TRAMP
  • v-FLIP TRAIL-FLIP
  • heterologous molecules are any member of the TNF family.
  • the polynucleotides of the invention encode a polypeptide which demonstrates a TRl 1, TRl ISVl and/or TRl 1SV2 functional activity.
  • a polypeptide demonstrating "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length and/or secreted TRl 1, TRl ISVl and/or TRl 1SV2 polypeptide.
  • Such functional activities include, but are not limited to, biological activity (e.g., ability to regulate (i.e., stimulate or inhibit) B cell proliferation (e.g., see Example 31), differentiation, activation, and or survival), antigenicity [ability to bind (or compete with a TRl 1, TRl ISVl and/or TRl 1SV2 polypeptide for binding) to an anti-TRl 1 antibody, anti- TRl ISVl antibody and/or anti-TRl 1SV2 antibody], immunogenicity (ability to generate antibody which binds to a TRl 1, TRl ISVl and/or TRl 1SV2 polypeptide), ability to form multimers with TRll, TRl lSVl and or TR11SV2 polypeptides of the invention, and ability to bind to a receptor or ligand for a TRl 1, TRl ISVl and/or
  • biological activity e.g., ability to regulate (i.e., stimulate or inhibit) B cell proliferation (e.g., see Example 31), differentiation, activation, and or survival
  • TR11SV2 e.g., Endokine-alpha (See, International Publication No. WO 98/07880 and Example 28)).
  • TRl 1 , TRl ISVl and/or TRl 1SV2 polypeptides, and fragments, variants, derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assay
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromotography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123.
  • physiological correlates of TRl 1, TRl ISVl and/or TRl 1SV2 binding to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of TRll, TRl ISVl and/or TR11SV2 polypeptides and fragments, variants derivatives and analogs thereof to elicit TRl 1, TRl ISVl and/or TRl 1SV2 related biological activity (e.g., to stimulate, or alternatively to inhibit (in the case of TRl l, TRl lSVl and/or TR11SV2 antagonists) B cell and/or T cell proliferation, differentiation, activation, and/or survival, in vitro or in vivo).
  • TRll, TRllSVl and/or TRl 1SV2 polypeptides of the invention are known to the skilled artisan and are within the scope of the invention.
  • polypeptides of the present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and contained within a recombinant host cell would be considered “isolated” for purposes of the present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host are polypeptides that have been purified, partially or substantially, from a recombinant host.
  • recombinantly produced versions of the TRl l, TRl lSVl, and TRl 1SV2 receptors can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • the polypeptides of the present invention also include: (a) the TRl 1 polypeptide encoded by the deposited cDNA including the leader; (b) the TRl ISVl polypeptide encoded by the deposited cDNA including the leader; (c) the TRl 1SV2 polypeptide encoded by the deposited cDNA including the leader; (d) the TRl 1 polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein); (e) the TRl ISVl polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein); (f) the TRl 1SV2 polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein); (g) the TRl 1 polypeptide of Figures 1 A and IB (SEQ ID NO:2) including the leader; (h) the TRl 1 polypeptide of Figures 1 A and IB (SEQ ID NO:2) including the leader; (h) the TR
  • TRl ISVl polypeptide of Figures 2A and 2B (SEQ ID NO:4) including the leader; (i) the TR11SV2 polypeptide of Figures 3A and 3B (SEQ ID NO:6) including the leader; (j) the TRl 1 polypeptide of Figures 1A and IB (SEQ ID NO:2) including the leader but minus the N-terminal methionine; (k) the TRl ISVl polypeptide of Figures 2A and 2B (SEQ ID NO:4) including the leader but minus the N-terminal methionine; (1) the
  • TR11SV2 polypeptide of Figures 3A and 3B (SEQ ID NO:6) including the leader but minus the N-terminal methionine; (m) the polypeptide of Figures 1A and IB (SEQ ID NO:2) minus the leader; (n) the polypeptide of Figures 2A and 2B (SEQ ID NO:4) minus the leader; (o) the polypeptide of Figures 3 A and 3B (SEQ ED NO:6) minus the leader; (p) the extracellular domain, the transmembrane domain, and the intracellular domain of the TRl 1 receptor shown in Figures 1A and IB (SEQ ID NO:2); (q) the extracellular domain, the transmembrane domain, and the intracellular domain of the TRl ISVl receptor shown in Figures 2A and 2B (SEQ ID NO:4); (r) the extracellular domain, the transmembrane domain, and the intracellular domain of the TRl 1SV2 receptor shown in Figures 3A and 3B (SEQ ID NO:
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TRl 1, TRl ISVl or TRl 1SV2 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of a TRl 1, TRl ISVl or TRl 1SV2 receptor.
  • a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figures 1A and IB (SEQ ED NO:2), Figures 2A and 2B (SEQ ID NO:4), and or Figures 3A and 3B (SEQ ID NO:6), the amino acid sequence encoded by deposited cDNA clones HHEAC71, HT5EA78, and HCFAZ22, respectively, or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C- termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TRl 1 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Arg-2 to about Pro-11 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-18 to about Arg-26 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Arg-34 to about Cys-42 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Arg-31 to about Glu-39 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gly-38 to about Asp-46 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gly-74 to about Ser-82 in S
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TRl ISVl receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ala-2 to about He- 10 in SEQ ID NO:4; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Asn-11 to about Gly-19 in SEQ ID NO:4; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-27 to about Ser-35 in SEQ ID NO:4; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Trp-38 to about Glu-46 in SEQ ID NO:4; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gly-42 to about Ser-50 in SEQ ID NO:4; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-31 to about Glu-46 in S
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TRl 1SV2 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gln-1 to about Cys-9 in SEQ ID NO:6; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gly-5 to about Arg- 13 in SEQ ID NO:6; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-18 to about Arg-26 in SEQ ID NO:6; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-29 to about Pro-37 in SEQ ID NO:6; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Cys-48 to about Glu-56 in SEQ ED NO:6;
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA S2:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
  • SMPS Simultaneous Multiple Peptide Synthesis
  • TRll, TRllSVl, TR11SV2 polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with heterologous polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • CH2, CH3 any combination thereof, including both entire domains and portions thereof
  • TRl 1 -immunoglobulin fusion polypeptides of the invention comprise, or altematively, consist of, amino acids -25 to 139, 1 to 139, 5 to 139, 1 to 130, 1 to 120, or 1 to 110, of SEQ ID NO:2 fused to an Fc domain.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ED NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 209341 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ED NO: 1 or contained in ATCC deposit No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ED NO:l), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:4, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 209342 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:3 or contained in ATCC deposit No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:3), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:6, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 209343 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:5 or contained in ATCC deposit No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:5), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as 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 the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more 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, and, most preferably, between about 15 to about 30 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, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
  • Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may 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, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985).
  • animals may be immunized with free peptide; however, anti -peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such aglutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti- peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813).
  • antigens e.g., insulin
  • FcRn binding partner such as IgG or Fc fragments
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958- 3964 (1995).
  • Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • the TR 11 , TR 11 S V 1 , and/or TR 11 S V2 polypeptides of the present invention and the epitope-bearing fragments thereof are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid).
  • a heterologous antigen e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid.
  • the heterologous antigen is an immunogen.
  • the heterologous antigen is the gpl20 protein of
  • HIV or a fragment thereof.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the TR 11 , TR 11 S V 1 , and/or TR 11 S V2 polypeptides of the present invention and the epitope-bearing fragments thereof are fused with polypeptide sequences of another TNF family member (or biologically active fragments or vari ants thereof) .
  • the TR 11 , TR 11 S V 1 , and or TR 11 S V2 polypeptides of the present invention are fused with a CD40L polypeptide sequence.
  • the CD40L polypeptide sequence is soluble.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to SEQ ID NO:l, SEQ ID NO:3, and/or SEQ ED NO:5, and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides of the invention are fusedwith soluble CD40L polypeptides, or biologically acitve fragments or variants thereof.
  • polypeptides of the present invention have uses, which include, but are not lmited to, as molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
  • TCR T-cell antigen receptors
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Immunoglobulins may have both a heavy and light chain.
  • An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos.
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded.
  • the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.
  • Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
  • Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions as described herein.
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 " 2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 “4 M, 10 " M, 5 X 10 “5 M, 10 “5 M, 5 X 10 ° M, 10 " 6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 " M, 10 “ 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 lO 15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
  • the invention features both receptor- specific antibodies and ligand-specific antibodies.
  • the invention also features receptor- specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.
  • receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • phosphorylation e.g., tyrosine or serine/threonine
  • antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
  • the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a "monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
  • Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples (e.g., Example 9).
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177- 186 (1995); Kettleborough et al., Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • chimeric, humanized, or human antibodies For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; 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 shuffling (U.S. Patent No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment, detection, and/or prevention in human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, 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 of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437- 444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and or binding domain and, as a consequence, bind to and neutralize polypeptide and or its ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and or to bind its ligands/receptors, and thereby block its biological activity.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2, and/or the amino acid sequence of SEQ ID NO:4, and/or the amino acid sequence of SEQ ID NO:6.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine rnAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242: 1038- 1041 (1988)).
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant vims expression vectors (e.g., cauliflower mosaic vims, CaMV; tobacco mosaic vims, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promote
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovims is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa califomica nuclear polyhedrosis vims (AcNPV) is used as a vector to express foreign genes.
  • the vims grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the vims and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovims transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant vims that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
  • the host cell may 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 identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al, J. Immunol.
  • polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2, a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:4, and/or a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:6, may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
  • polypeptides corresponding to SEQ ID NO:2 may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
  • the polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins, such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci.
  • hexahistidine provides for convenient purification of the fusion protein.
  • Other peptide tags useful 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 present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
  • the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 I, ⁇ n In or "Tc.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxombicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunombicin (formerly daunomycin) and doxombicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincris
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the d g moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an a
  • VEGI See, International Publication No. WO 99/23105
  • CD40 Ligand a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, 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.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • the antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples.
  • the translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types.
  • Monoclonal antibodies directed against a specific epitope, or combination of epitopes will allow for the screening of cellular populations expressing the marker.
  • Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al, Cell, 96:737-49 (1999)).
  • hematological malignancies i.e. minimal residual disease (MRD) in acute leukemic patients
  • GVHD Graft-versus-Host Disease
  • these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
  • the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate.at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium de
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS- PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off -rate of an antibody- antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off -rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
  • the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating, detecting, and/or preventing one or more of the disclosed diseases, disorders, or conditions.
  • Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
  • the antibodies of the invention can be used to treat, diagnose, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein (e.g., autoimmune diseases, disorders, or conditions associated with such diseases or disorders, including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter
  • antibodies of the invention are be used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.
  • antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis.
  • the treatment, detection, and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents, antibiotics, and immunoglobulin). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred.
  • human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
  • polypeptides or polynucleotides of the present invention It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention.
  • Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 "4 M, 10 “4 M, 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 X 10 “8 M, 10 “8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 " M, 10 11 M, 5 X 10 "12 M, 10 12 M, 5 X 10 " ' 3 M, 10 " 13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 15 M, and 10 "15 M.
  • nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
  • the nucleic acids produce their encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
  • nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
  • nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad.
  • the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
  • Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc.
  • nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to dismpt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad.
  • viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
  • a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
  • the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al.,
  • Adenovimses are other viral vectors that can be used in gene therapy. Adenovimses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenovimses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenovimses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
  • adenovims vectors are used.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618- 644 (1993); Cline, Pharmac. Ther.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • Recombinant blood cells are preferably administered intravenously.
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
  • the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
  • in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
  • the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
  • the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
  • a compound 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, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
  • the compound or composition can be delivered in a controlled release system.
  • a pump may 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)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Dmg Bioavailability, Drug Product Design and Performance, 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., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constmcting it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • the present invention also provides pharmaceutical compositions.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be 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 and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of 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 vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Diagnosis and Imaging Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention.
  • the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
  • the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 131 I, 125 I, 123 I, ,21 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( u5m In, 113m In, 112 In, m In), and technetium ("Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum (“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, ,86 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
  • Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
  • specific embodiments of the invention are directed to the use of the antibodies of the invention to quantitate or qualitate concentrations of cells of B cell lineage or cells of monocytic lineage.
  • antibodies of the invention may be used to treat, diagnose, or prognose an individual having an immunodeficiency.
  • antibodies of the invention are used to treat, diagnose, and/or prognose an individual having common variable immunodeficiency disease (CVID) or a subset of this disease.
  • CVID common variable immunodeficiency disease
  • antibodies of the invention are used to diagnose, prognose, treat or prevent a disorder characterized by deficient serium immunoglobulin production, recurrent infections, and/or immune system dysfunction.
  • antibodies of the invention may be used to treat, diagnose, or prognose an individual having an autoimmune disease or disorder.
  • antibodies of the invention are used to treat, diagnose, and/or prognose an individual having systemic lupus erythematosus, or a subset of the disease.
  • antibodies of the invention are used to treat, diagnose and or prognose an individual having rheumatoid arthritis, or a subset of this disease.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99n Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
  • monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050).
  • the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instmment.
  • the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
  • the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • kits that can be used in the above methods.
  • a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
  • the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
  • the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
  • the kits of the present invention comprise two or more antibodies (monoclonal and/or polyclonal) that recognize the same and/or different sequences or regions of the polypeptide of the invention.
  • kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (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, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
  • a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
  • the kit is a diagnostic kit for use in screening serum containing antibodies specific against prohferative and/or cancerous polynucleotides and polypeptides.
  • a kit may include a control antibody that does not react with the polypeptide of interest.
  • a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
  • a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry).
  • the kit may include a recombinantly produced or chemically synthesized polypeptide antigen.
  • the polypeptide antigen of the kit may also be attached to a solid support.
  • the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
  • a kit may also include a non-attached reporter-labeled anti-human antibody.
  • binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
  • the invention includes a diagnostic kit for use in screening semm containing antigens of the polypeptide of the invention.
  • the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
  • the antibody is attached to a solid support.
  • the antibody may be a monoclonal antibody.
  • the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
  • the reagent After binding with specific antigen antibody to the reagent and removing unbound semm components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti- antigen antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include nonspecific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s). Thus, the invention provides an assay system or kit for carrying out this diagnostic method.
  • the kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface- bound anti-antigen antibody.
  • the invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies that disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Included are both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies that activate the receptor may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.
  • the antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • antibodies that bind to TRl 1, TRl ISVl , and or TRl 1SV2 irrespective of whether TRl 1, TRl ISVl, and/or TRl 1SV2 is bound to a TRl 1, TRllSVl, and/or TRl 1SV2 ligand.
  • These antibodies act as TRll, TRllSVl, and/or TRl 1SV2 agonists as reflected in an increase in cellular proliferation in response to binding of TRl l, TRl lSVl, and/or TRl 1SV2 to a TRll, TRllSVl, and/or TRl 1SV2 ligand in the presence of these antibodies.
  • the above antibody agonists can be made using methods known in the art.
  • the invention encompasses antibodies that inhibit or reduce the ability of TRl 1 , TRllSVl, and/or TRl 1SV2 to bind TRll, TRl lSVl, and or TRl 1SV2 ligand in vitro and/or in vivo.
  • antibodies of the invention inhibit or reduce the ability of TRl l, TRl lSVl, and/or TRl 1SV2 to bind TRl l, TRllSVl, and/or TR11SV2 ligand in vitro.
  • antibodies of the invention inhibit or reduce the ability of TRl 1, TRl ISVl, and/or TRl 1SV2 to bind bind TRl l, TRl lSVl, and/or TR11SV2 ligand in vivo. Such inhibition can be assayed using techniques described herein or otherwise known in the art.
  • the invention also encompasses, antibodies that bind specifically to TRl 1, TRllSVl, and/or TRl 1SV2, but do not inhibit the ability of TRll, TRl lSVl, and/or TR11SV2 to bind TRl l, TRl lSVl, and/or TRl 1SV2 ligand in vitro and/or in vivo.
  • antibodies of the invention do not inhibit or reduce the ability of TRl 1, TRl ISVl, and/or TRl 1SV2 to bind TRl 1, TRl ISVl, and/or TRl 1SV2 ligand in vitro.
  • antibodies of the invention do not inhibit or reduce the ability of TRll, TRl lSVl, and/or TR11SV2 to bind TRl l, TRl lSVl, and/or TRl 1SV2 ligand in vivo.
  • the invention encompasses antibodies that inhibit or reduce a TRl 1-, TRl 1SV1-, and or TRl lSV2-mediated biological activity in vitro and/or in vivo.
  • antibodies of the invention inhibit or reduce TRl 1, TRl ISVl, and or TRl lSV2-mediated B or T cell proliferation in vitro. Such inhibition can be assayed by routinely modifying B or T cell proliferation assays described herein or otherwise known in the art.
  • antibodies of the invention inhibit or reduce TRl 1-, TRl 1SV1-, and or TRl 1SV2- mediated B or T cell proliferation in vivo.
  • the invention also encompasses, antibodies that bind specifically to a TRl 1, TRl ISVl, and/or TRl 1SV2, but do not inhibit or reduce a TRl 1-, TRl 1SV1-, and or TRl lSV2-mediated biological activity in vitro and/or in vivo (e.g., stimulation of B or T cell proliferation).
  • antibodies of the invention do not inhibit or reduce a TRl 1-, TRl 1SV1-, and/or TRl lSV2-mediated biological activity in vitro.
  • antibodies of the invention do not inhibit or reduce a TRl 1-, TRl 1SV1-, and/or TRl lSV2-mediated biological activity in vivo.
  • the invention encompasses antibodies that specifically bind to the same epitope as at least one of the antibodies specifically referred to herein, in vitro and/or in vivo.
  • the specific antibodies described above are humanized using techniques described herein or otherwise known in the art and then used as therapeutics as described herein. In another specific embodiment, any of the antibodies listed above are used in a soluble form.
  • any of the antibodies listed above are conjugated to a toxin or a label (as described infra). Such conjugated antibodies are used to kill a particular population of cells or to quantitate a particular population of cells. In a preferred embodiment, such conjugated antibodies are used to kill B cells expressing TRll, TRllSVl, and/or TR11SV2 on their surface. In another preferred embodiment, such conjugated antibodies are used to quantitate B cells expressing TRl 1, TRl ISVl, and/or TRl 1SV2 on their surface. In a preferred embodiment, such conjugated antibodies are used to kill T cells expressing TRl 1, TRl ISVl, and/or TRl 1SV2 on their surface. In another preferred embodiment, such conjugated antibodies are used to quantitate T cells expressing TRl 1, TRl ISVl, and/or TRl 1SV2 on their surface.
  • any of the antibodies listed above are conjugated to a toxin or a label (as described infra). Such conjugated antibodies are used to kill a particular population of cells or to quantitate a particular population of cells.
  • the antibodies of the invention also have uses as therapeutics and/or prophylactics which include, but are not limited to, inactivating lymphocytes or blocking lymphocyte activation and/or killing lymphocyte lineages that express TRll, TRl ISVl, and/or TRl 1SV2 on their cell surfaces (e.g., to treat, prevent, and/or diagnose myeloid leukemias, lymphocyte based leukemias and lymphomas, lymphocytosis, lymphocytopenia, rheumatoid arthritis, and other diseases or conditions associated with activated lymphocytes).
  • the antibodies of the invention fix complement.
  • the antibodies of the invention are associated with heterologous polypeptides or nucleic acids (e.g. toxins, such as, compounds that bind and activate endogenous cytotoxic effecter systems, and radioisotopes; and cytotoxic prodrugs).
  • heterologous polypeptides or nucleic acids e.g. toxins, such as, compounds that bind and activate endogenous cytotoxic effecter systems, and radioisotopes; and cytotoxic prodrugs.
  • one or more monoclonal antibodies are produced wherein they recognize or bind TRl 1 , TRl 1SV 1 , and/or TRl 1SV2 and/or a mutein thereof, but do not recognize or bind TRl l, TRl lSVl, and/or TRl 1SV2 and/or a mutein thereof.
  • one or more monoclonal antibodies are produced wherein they recognize or bind TRl 1, TRl ISVl, and/or TRl 1SV2 and/or a mutein thereof, but do not recognize or bind TRl 1, TRl ISVl, and/or TRl 1SV2 and/or a mutein thereof.
  • antibodies to the TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the TRl 1, TRl ISVl, and or TRl 1SV2, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437- 444 (1989), and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to TRl l, TRl lSVl, and or TRl 1SV2 and competitively inhibit TRl l, TRl lSVl, and/or TR11SV2 multimerization and/or binding to ligand can be used to generate anti-idiotypes that "mimic" the TRl 1, TRl ISVl, and/or TRl 1SV2 TNF mutimerization and or binding domain and, as a consequence, bind to and neutralize TRl l, TRl lSVl, and/or TR11SV2 and or its ligand.
  • Such neutralizing anti- idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize TRl 1, TRl ISVl, and/or TRl 1SV2 ligand.
  • anti-idiotypic antibodies can be used to bind TRl 1 , TRl ISVl , and or TRl 1SV2 on the surface of cells of B or T cell lineage, and thereby block TRl 1-, TRl 1SV1-, and/or TRl 1SV2- mediated B or T cell activation, proliferation, and/or differentiation.
  • TNF-family ligands induce various cellular responses by binding to TNF-family ligands
  • TNF-family receptors including the TRl 1, TRl ISVl, and TRl 1SV2 receptors of the present invention.
  • TNF-beta a potent ligand of the TNF receptor proteins, is known to be involved in a number of biological processes including lymphocyte development, tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle and Homer, Prog. Allergy, 40:162-182 (1988)).
  • TNF-alpha also a ligand of the TNF receptor proteins, has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al, J. Immunol. 136(7):2483 (1987); Porter, Tibtech 9:158-162 (1991)), growth regulation, vascular endothelium effects and metabolic effects.
  • TNF-alpha also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation.
  • TNF-alpha up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1.
  • TNF-alpha and the Fas ligand have also been shown to induce programmed cell death.
  • TRAIL also known as Apo-2L
  • TNF tumor necrosis factor
  • the human receptor for TRAIL was found to be an undescribed member of the TNF receptor family designated death receptor (DR)-4 (Pan, G., et al, Science 276:111-113 (1997)).
  • Cells which express the TRl 1, TRl ISVl or TRl 1SV2 polypeptides and are believed to have a potent cellular response to TRl 1, TRl ISVl or TRl 1SV2 receptor ligands include activated T-cells.
  • a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
  • such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis.
  • Apoptosis-programmed cell death-is a physiological mechanism involved in the deletion of peripheral T-lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes (Ameisen, J.C., AIDS 8: 1197-1213 (1994); Krammer, P.H. et al., Curr. Opin. Immunol. 6:279-289 (1994)).
  • TRll, TRl ISVl, and TRl 1SV2 receptor proteins and mRNAs encoding the TRl 1, TRl ISVl, and TRl 1SV2 receptor proteins when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the disease state.
  • body fluids e.g., sera, plasma, urine, and spinal fluid
  • the invention provides a diagnostic method useful during diagnosis of disease states, which involves assaying the expression level of the gene encoding the TRl 1, TRl ISVl, and TRl 1SV2 receptor proteins in mammalian cells or body fluid and comparing the gene expression level with a standard TRl 1, TRl ISVl, and
  • TRl 1SV2 receptor gene expression levels whereby an increase or decrease in the gene expression level over the standard is indicative of certain disease states associated with aberrant cell survival.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting significantly aberrant TRl 1, TRl ISVl or TRl 1SV2 receptor gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.
  • test the expression level of the gene encoding the TRll, TRl lSVl or TRl 1SV2 receptor protein is intended qualitatively or quantitatively measuring or estimating the level of the TRl 1, TRl ISVl or TRl 1SV2 receptor protein or the level of the mRNA encoding the TRl 1, TRl ISVl or TRl 1SV2 receptor protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the TRl 1, TRl ISVl or TRl 1SV2 receptor protein level or mRNA level in a second biological sample).
  • the TRl 1, TRl ISVl or TRl 1SV2 receptor protein levels or mRNA levels in the first biological sample is measured or estimated and compared to a standard TRl 1, TRl ISVl or TRl 1SV2 receptor protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disease state.
  • a standard TRl 1, TRl ISVl or TRl 1SV2 receptor protein level or mRNA level the standard being taken from a second biological sample obtained from an individual not having the disease state.
  • TRl 1SV2 receptor protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains TRl 1, TRl ISVl or TRl 1SV2 receptor protein or mRNA.
  • Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature TRl 1, TRl ISVl or TRl 1SV2 receptor protein, and thymus, prostate, heart, placenta, muscle, liver, spleen, lung, kidney and other tissues. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • TRl 1, TRl ISVl or TRl 1SV2 receptor polynucleotides and/or polypeptides of the invention regulate cell survival and/or proliferation.
  • Diseases associated with increased cell survival, or the inhibition of apoptosis, that may be treated, detected or prevented with the polypeptides or polynucleotides of the invention, or agonists or antagonists thereof include, but are not limited to, cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides, polypeptides, agonists, or antagonists of the invention are used to diagnose and/or prevent growth, progression, and/or metastasis of cancers, in particular those listed above or in the paragraph that follows.
  • Additional diseases or conditions assocaiated with increased cell survival that may be treated, detected or prevented with the polypeptides or polynucleotides of the invention, or agonists or antagonists thereof include, but are not limited to, progression, and or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to
  • TRl 1, TRl 1SV 1 , TRl 1SV2 polynucleotides or polypeptides of the invention are used to treat, detect or prevent autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas.
  • lymphocytic leukemias including, for example, MLL and chronic lymphocytic leukemia (CLL)
  • CLL chronic lymphocytic leukemia
  • TRl 1, TRl ISVl, TRl 1SV2 polynucleotides or polypeptides of the invention are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
  • cancerous cells or tissue e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma
  • cancer therapy e.g., chemotherapy or radiation therapy
  • Diseases associated with increased apoptosis that may be treated, detected or prevented with the polypeptides or polynucleotides of the invention, or agonists or antagonists thereof, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Grave's disease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, thrombocytopenic purpura, and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft vs.
  • TRl l, TRl ISV l or TRHSV2 polynucleotides, polypeptides, agonists, or antagonists of the invention are used to diagnose, prevent, and/or treat the diseases and disorders listed above.
  • Immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed with TRl 1, TRl ISVl, and/or TRl 1SV2 polynucleotides or polypeptides or TRl 1, TRl ISVl, and/or TRl 1SV2 agonists or antagonists (e.g., anti-TRl 1, anti- TRl ISV l, and/or anti-TRl 1SV2 antibodies) of the invention, include, but are not limited to one or more immunodeficiencies selected from: severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), DiGeorge anomaly, Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-
  • Wiskott-Aldrich Syndrome WAS
  • X-linked immunodeficiency with hyper IgM non X-linked immunodeficiency with hyper IgM
  • selective IgA deficiency IgG subclass deficiency (with or without IgA deficiency)
  • antibody deficiency with normal or elevated Igs immunodeficiency with thymoma
  • Ig heavy chain deletions Ig heavy chain deletions
  • kappa chain deficiency B cell lymphoproliferative disorder (BLPD)
  • selective IgM immunodeficiency recessive agammaglobulinemia (Swiss type)
  • reticular dysgenesis neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism
  • XLP X- linked lymphoprolifer
  • Autoimmune diseases or disorders that may be treated, diagnosed, or prognosed using TRl 1 , TRl 1SV 1 , and/or TRl 1SV2 polynucleotides or polypeptides or TRl 1 , TRl ISVl, and/or TRl 1SV2 agonists or antagonists (e.g., anti-TRl 1, anti-TRl ISVl, and/or anti-TRl 1SV2 antibodies) of the invention include, but are not limited to, one or more of the following: autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthal
  • TRl 1 , TRl ISVl, and/or TRl 1SV2 polynucleotides or polypeptides of the invention, or agonists or antagonists thereof may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post- transfusion purpura.
  • diseases or disorders, or conditions associated therewith include primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection),
  • TRl l, TRl lSVl, and/or TR11SV2 polynucleotides or polypeptides of the invention, or agonists or antagonists thereof may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable mutliple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g, neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g, cytamegalovims (CMV)-negative recipients of CMN-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (CMV
  • an animal e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human
  • boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides of the invention, and/or agonists thereof are administered to boost the immune system to produce increased quantities of IgG.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides of the invention and/or agonists thereof are administered to boost the immune system to produce increased quantities of IgA.
  • TRll, TRl lSVl, and/or TR11SV2 polypeptides of the invention and/or agonists thereof are administered to boost the immune system to produce increased quantities of IgM.
  • Assays available to detect levels of soluble receptors are well known to those of skill in the art, for example, radioimmunoassays, competitive-binding assays, Western blot analysis, and preferably an ELISA assay may be employed.
  • the present invention is directed to a method for inhibiting an activity of TRl 1, TRl ISVl or TRl 1SV2 induced by a TNF-family ligand (e.g., cell proliferation, hematopoietic development), which involves administering to a cell which expresses a TRl 1, TRl ISVl or TRl 1SV2 polypeptide an effective amount of a TRl 1, TRl ISVl or TRl 1SV2 receptor ligand, analog or an antagonist capable of decreasing TRl 1, TRl ISVl or TRl 1SV2, receptor mediated signaling.
  • TRl 1 e.g., TRl 1, TRl ISVl or TRl 1SV2 induced by a TNF-family ligand (e.g., cell proliferation, hematopoietic development)
  • TRl 1 e.g., cell proliferation, hematopoietic development
  • TRl ISVl or TRl 1SV2 receptor mediated signaling is increased to treat, detect, and/or prevent a disease wherein increased cell proliferation is exhibited.
  • An antagonist can include soluble forms of the TRl 1, TRl ISVl or TRl 1SV2 receptors and antibodies directed against the TRll, TRllSVl or TRl IS V2 polypeptides which block TRll, TRl ISVl or TRl 1SV2 receptor mediated signaling.
  • TRl 1, TRl ISVl or TRl 1SV2 receptor mediated signaling is decreased to treat, detect, and or prevent a disease.
  • the present invention is directed to a method for increasing cell proliferation induced by a TNF-family ligand, which involves administering to a cell which expresses a TRl 1, TRl ISVl or TRl 1SV2 polypeptide an effective amount of an agonist capable of increasing TRl 1 , TRl ISVl or TRl 1SV2 receptor mediated signaling.
  • TRl 1 , TR 11 SV 1 or TRl 1 SV2 receptor mediated signaling is increased to treat, detect, and/or prevent a disease wherein decreased cell proliferation is exhibited.
  • Agonists of the present invention include monoclonal antibodies directed against the TR 11 , TR USVl or TRll SV2 polypeptides which stimulate TRl 1, TRl ISVl or TRl 1SV2 receptor mediated signaling.
  • TRl 1, TRl ISVl or TRl 1SV2 receptor mediated signaling is increased to treat, detect, and/or prevent a disease.
  • agonist is intended naturally occurring and synthetic compounds capable of enhancing cell proliferation and differentiation mediated by TRll, TRllSVl or TR11SV2 polypeptides.
  • Such agonists include agents which increase expression of TRl 1, TRl ISVl or TRl 1SV2 receptors or increase the sensitivity of the expressed receptor.
  • antagonists include agents which decrease expression of TRll, TRl ISVl or TRl 1SV2 receptors or decrease the sensitivity of the expressed receptor.
  • One such screening technique involves the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science 246:181-296 (October 1989).
  • compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g., signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
  • Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the receptor.
  • the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
  • Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention -antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof.
  • Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand.
  • the ligand can be labeled, e.g., by radioactivity.
  • the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
  • Soluble forms of the polypeptides of the present invention may be utilized in the ligand binding assay described above. These forms of the TRll, TRllSVl, and TRl 1SV2 receptors are contacted with ligands in the extracellular medium after they are secreted. A determination is then made as to whether the secreted protein will bind to TRl 1, TRl ISVl or TRl 1SV2 receptor ligands. Further screening assays for agonist and antagonist of the present invention are described in Tartaglia, L.A., and Goeddel, D.V., J. Biol. Chem. 267(7):4304- 4307(1992).
  • a screening method for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.
  • the method involves contacting cells which express TRl 1 , TRl ISVl or TRl 1SV2 polypeptides with a candidate compound and a TNF- family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway.
  • assaying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF- family ligand (e.g., determining or estimating an increase or decrease in T cell proliferation or tritiated thymidine labeling).
  • a cell expressing a TRll, TRllSVl or TR11SV2 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
  • a thymocyte proliferation assay may be employed to identify both ligands and potential dmg candidates. For example, thymus cells are disaggregated from tissue and grown in culture medium.
  • Agonists according to the present invention include compounds such as, for example, TNF-family ligand peptide fragments, transforming growth factors, and neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate).
  • Preferred agonists include polyclonal and monoclonal antibodies raised against TRl 1, TRl ISVl or TRl 1SV2 polypeptides, or a fragments thereof.
  • Such agonist antibodies raised against a TNF-family receptor are disclosed in Tartaglia, L.A., et al, Proc. Natl. Acad. Sci. USA SS:9292-9296 (1991); and Tartaglia, L.A., and Goeddel, D.V., J Biol.
  • chemotherapeutic drugs such as, for example, cisplatin, doxombicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and amyloid peptide. (Science 267:1457-1458 (1995)).
  • antagonists according to the present invention are nucleic acids corresponding to the sequences contained in TRll, TRllSVl and/or TRl 1SV2, or the complementary strand thereof, and/or to nucleotide sequences contained in the deposited clones HHEAC71, HCFAZ22, and HT5EA78, respectively.
  • antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
  • Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation.
  • Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
  • Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 10-1573 (1979); Cooney et al, Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991).
  • the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
  • the TRl 1, TRl ISVl and/or TRl 1SV2 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the TRl 1, TRl ISVl and/or TRl 1SV2 antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding TRl 1, TRl ISVl and/or TRl 1SV2, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive.
  • Such promoters include, but are not limited to, the SV40 early promoter region (Bemoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a TRl 1, TRl ISVl and or TRl 1SV2 gene.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded TRl 1, TRl ISVl and/or TRl 1SV2 antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a TRl 1, TRl ISVl and/or TRl 1SV2 RNA it may contain and still form a stable duplex (or triplex as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Oligonucleotides that are complementary to the 5' end of the message, e.g., the
  • oligonucleotides complementary to either the 5'- or 3'- non-translated, non- coding regions of the TRl 1, TRl ISVl and/or TRl 1SV2 shown in Figures 1A-B, 2A-B, and 3A-B, respectively, could be used in an antisense approach to inhibit translation of endogenous TRl 1, TRl ISVl and or TRl 1SV2 mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of TRl l, TRl lSVl and/or TR11SV2 mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • the polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosyl
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an a-anomeric oligonucleotide.
  • An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2 ⁇ -0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).
  • Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • TRl 1SV2 coding region sequences could be used, those complementary to the transcribed untranslated region are most preferred.
  • Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science 247: 1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy TRll, TRllSVl and or TRl 1SV2 mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • hammerhead ribozymes The constmction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).
  • TRll Trigger-activated ribozyme
  • TRl Trigger-activated ribozyme
  • TRl 1SV2 Trigger-activated ribozyme
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the TRl 1, TRl ISVl and/or TRl 1SV2 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express TRl 1, TRl ISVl and or TRl 1SV2 in vivo.
  • DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous TRl 1 , TRl ISVl and or TRl 1SV2 messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous gene expression can also be reduced by inactivating or "knocking out" the TR 11 , TR 11 S V 1 and or TR 11 S V2 gene and/or its promoter using targeted homologous recombination.
  • endogenous gene expression can also be reduced by inactivating or "knocking out" the TR 11 , TR 11 S V 1 and or TR 11 S V2 gene and/or its promoter using targeted homologous recombination.
  • a mutant, non-functional polynucleotide of the invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo.
  • techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene.
  • Antagonists according to the present invention include soluble forms of the TRl l, TRllSVl, and TRHSV2 receptors (e.g., fragments of the TRl l, TRl lSVl, and TRl 1SV2 receptors shown in Figures 1 A and IB, 2 A and 2B, and 3 A and 3B, respectively, that include the ligand binding domain from the extracellular region of the full length receptor).
  • soluble forms of the receptor which may be naturally occurring or synthetic, antagonize TRl l, TRllSVl, and TRHSV2 mediated signaling by competing with the cell surface bound forms of the receptor for binding to TNF- family ligands.
  • TRl 1-Fc fusion protein containing the extracellular domain of TRl 1 has been found by the inventors to inhibit B cell proliferation (data not shown).
  • Antagonists of the present invention also include antibodies specific for TNF- family ligands and TRl 1-, TRl 1SV1-, and TRl lSV2-Fc fusion proteins.
  • TNF-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing the ligand receptor signaling pathway.
  • Members of the TNF ligand family include, but are not limited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med.
  • endokine-alpha International Publication No. WO 98/07880
  • TR6 International Publication No. WO 98/30694
  • OPG and neutrokine-alpha
  • neutrokine-alpha International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF)
  • soluble forms of Fas CD30, CD27, CD40 and 4-IBB
  • DR3 International Publication No. WO 97/33904
  • DR4 International Publication No. WO 98/32856
  • TR5 International Publication No. WO 98/30693
  • TR6 International Publication No. WO 98/30694
  • TR7 International Publication No.
  • WO 98/41629 TRANK
  • TR9 International Publication No. WO 98/56892
  • TR10 International Publication No. WO 98/54202
  • 312C2 International Publication No. WO 98/06842
  • TR12 and soluble forms CD154, CD70, and CD153.
  • TNF-alpha has been shown to protect mice from infection with herpes simplex virus type 1 (HSV-1). Rossol-Voth, R. et al, J .Gen. Virol. 72:143-147 (1991). The mechanism of the protective effect of TNF-alpha is unknown but appears to involve neither interferons not NK cell killing.
  • One member of the TNFR family has been shown to mediate HSV-1 entry into cells. Montgomery, R. et al, Eur. Cytokine Newt. 7:159 (1996). Further, antibodies specific for the extracellular domain of this TNFR block HSV-1 entry into cells.
  • TRl 1, TRl ISVl, and TRl 1SV2 antagonists of the present invention include both TRl 1, TRl ISVl, and TRl 1SV2 amino acid sequences and antibodies capable of preventing TNFR mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized TNFR for binding to vims or by directly blocking binding of vims to cell surface receptors.
  • Antibodies according to the present invention may be prepared by any of a variety of methods using TRl l, TRl lSVl, and TR11SV2 receptor immunogens of the present invention.
  • TRl 1, TRl ISVl, and TRl 1SV2 receptor immunogens include the TRl 1, TRl ISVl, and TRl 1SV2 receptor proteins shown in Figures 1A and IB, 2A and 2B, and 3A and 3B (SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively; which may or may not include a leader sequence) and polypeptide fragments of the receptor comprising the ligand binding, extracellular, transmembrane, the intracellular domains of the TRl 1, TRl ISVl , and TRl 1SV2 receptors, or any combination thereof.
  • Antibody and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed in Tartaglia and Goeddel, J Biol. Chem. 267(7):4304-4307(1992)); Tartaglia et al, Cell 75:213-216 (1993)), and PCT Application WO 94/09137.
  • the term "antibody” (Ab) or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') fragments) which are capable of binding an antigen.
  • Fab and F(ab') fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)).
  • antibodies according to the present invention are mAbs.
  • Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody Technology," In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon et al, eds.), Elsevier, Amsterdam (1984)).
  • Proteins and other compounds which bind the TRl 1 , TRl ISVl , and TRl 1SV2 receptor domains are also candidate agonist and antagonist according to the present invention.
  • binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
  • yeast two-hybrid system Fields and Song, Nature 340:245-246 (1989)
  • a modified version of the yeast two-hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al., Cell 75:791-803 (1993); Zervos, A.S. et al., Cell 72:223-232 (1993)).
  • the yeast two-hybrid system is used according to the present invention to capture compounds which bind to the ligand binding, extracellular, intracellular, and transmembrane domains of the TRl 1, TRl ISVl, and TRl 1SV2 receptors.
  • Such compounds are good candidate agonist and antagonist of the present invention.
  • the intracellular domain of the TRl 1, TRl ISVl, and TRl 1SV2 receptors, or portions thereof may be used to identify cellular proteins which interact with the receptor in vivo.
  • Such an assay may also be used to identify ligands with potential agonistic or antagonistic activity of TRll, TRl lSVl, and TR11SV2 receptor function.
  • screening techniques include the use of cells which express the polypeptide of the present invention (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, 246:181-296 (1989).
  • potential agonists or antagonists may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, e.g., signal transduction may be measured to determine whether the potential antagonist or agonist is effective.
  • TNF Tumor Necrosis Factor
  • the Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D.V. et al, "Tumor Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant. Biol. 57:597-609 (1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev. Biochem. 57:505-518 (1988); Old, L.J., Sci. Am. 258:59-75 (1988); Fiers, W., EERS Lett. 285: 199-224 (1991)).
  • the TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.
  • TRl l, TRl IS VI, and TR11SV2 agonists or antagonists of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of TRl 1, TRl ISVl, and/or TRl 1SV2.
  • TRl 1, TRl lSVl, and TRl 1SV2 polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder.
  • a gene therapy approach may be applied to treat and/or prevent such disorders.
  • TRl l, TRl lSVl, and TR11SV2 nucleotide sequences permits the detection of defective TRl 1, TRl ISVl, and TRl 1SV2 genes, and the replacement thereof with normal TRll, TRllSVl, and TR11SV2 encoding genes.
  • Defective genes may be detected in in vitro diagnostic assays, and by comparision of the TRl 1, TRl ISVl, and TRl 1SV2 nucleotide sequence disclosed herein with that of a TRl 1, TRl ISVl, and TRl 1SV2 gene derived from a patient suspected of harboring a defect in this gene.
  • the polypeptides of the present invention are used as a research tool for studying the biological effects that result from inhibiting Endokine- alpha/ Endokine-alpha TRl 1, TRl ISVl, and/or TRl 1SV2 interactions on different cell types.
  • TRl 1, TRllSVl, and TR11SV2 polypeptides also may be employed in in vitro assays for detecting Endokine-alpha or TRll, TRl ISVl, and/or TRl 1SV2 or the interactions thereof.
  • a purified TRl 1, TRl ISVl, and/or TRl 1SV2 antagonist (including soluble TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides) is used to inhibit binding of Endokine-alpha to endogenous cell surface Endokine-alpha receptors.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 antagonist including soluble TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides
  • Certain ligands of the TNF family have been reported to bind to more than one distinct cell surface receptor protein.
  • soluble TRll, TRllSVl, and/or TR11SV2 polypeptides of the present invention may be employed to inhibit the binding of Endokine-alpha not only to cell surface TRl 1, TRl ISVl, and/or TRl 1SV2, but also Endokine-alpha receptor proteins that are distinct from TRl 1, TRl ISVl, and/or TRl 1SV2 antagonist (including soluble TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides).
  • TRl 1, TRl ISVl, and/or TRl 1SV2 antagonists of the invention is used to inhibit a biological activity of Endokine-alpha, in in vitro or in vivo procedures.
  • TRl 1, TRl ISVl, and/or TRl 1SV2 polypeptides is used to inhibit a biological activity of Endokine-alpha, in in vitro or in vivo procedures.
  • antagonists of the invention By inhibiting binding of Endokine-alpha to cell surface receptors, antagonists of the invention also inhibit biological effects that result from the binding of Endokine-alpha to endogenous receptors.
  • TRll, TRl lSVl, and/or TR11SV2 antagonists may be employed, including, for example, the above-described TRl 1, TRl ISVl, and/or TRl 1SV2 fragments, derivatives, and variants that are capable of binding Endokine- alpha.
  • TRl 1 , TRl ISVl , and/or TRl 1SV2 polypeptide of the invention is employed to inhibit a biological activity of Endokine- alpha.
  • a TRl 1, TRl ISVl, and/or TRl 1SV2 agonist or antagonist of the invention is administered to a mammal (e.g., a human) to treat, detect, and/or prevent an Endokine-alpha mediated disorder.
  • a mammal e.g., a human
  • Endokine-alpha mediated disorders include conditions caused (directly or indirectly) or exacerbated by
  • TRl l, TRl lSVl, and TRl 1SV2 receptor agonists may be employed to stimulate ligand activities, such as inhibition of tumor growth and necrosis of certain transplantable tumors.
  • the agonists may also be employed to stimulate cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation. Agonists to the TRl 1,
  • TRl lSVl, and TR11SV2 receptors may also augment the role of TRll, TRllSVl, and TRl 1SV2 in the host's defense against microorganisms and prevent related diseases (infections such as that from Listeria monocytogenes) and Chlamidiae.
  • the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.
  • Agonists to the receptor polypeptides of the present invention may be used to augment TNF's role in host defenses against microorganisms and prevent related diseases.
  • the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.
  • the agonists may also be employed to mediate an anti-viral response, to regulate growth, to mediate the immune response and to treat, detect, and/or prevent immunodeficiencies related to diseases such as HIV by increasing the rate of lymphocyte proliferation and differentiation.
  • the antagonists to the polypeptides of the present invention may be employed to inhibit ligand activities, such as stimulation of tumor growth and necrosis of certain transplantable tumors.
  • the antagonists may also be employed to inhibit cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation.
  • Antagonists may also be employed to treat, detect, and/or prevent autoimmune diseases, for example, graft versus host rejection and allograft rejection, and T-cell mediated autoimmune diseases such as AIDS. It has been shown that T-cell proliferation is stimulated via a type 2 TNF receptor. Accordingly, antagonizing the receptor may prevent the proliferation of T-cells and treat, detect, and/or prevent T-cell mediated autoimmune diseases.
  • HIV -induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J.C., AIDS 8: 1197-1213 (1994) ; Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol. 6:605-615(1995); Muro-Cacho, CA. et al, J. Immunol. 754:5555-5566 (1995)).
  • apoptosis and CD4+ T-lymphocyte depletion is tightly correlated in different animal models of AEDS (Brunner, T., et al, Nature 373:441-444 (1995); Gougeon, M.L., et al, AIDS Res. Hum. Retroviruses 9:553- 563 (1993)) and, apoptosis is not observed in those animal models in which viral replication does not result in AIDS (Gougeon, M.L. et al, AIDS Res. Hum. Retroviruses 9:553-563 (1993)).
  • TNF-family ligand was detectable in uninfected macrophages and its expression was upregulated following HIV infection resulting in selective killing of uninfected CD4+ T-lymphocytes (Badley, A.D et al, J. Virol. 70:199-206 (1996)).
  • the immune system of the recipient animal In rejection of an allograft, the immune system of the recipient animal has not previously been primed to respond because the immune system for the most part is only primed by environmental antigens. Tissues from other members of the same species have not been presented in the same way that, for example, vimses and bacteria have been presented.
  • immunosuppressive regimens are designed to prevent the immune system from reaching the effector stage.
  • the immune profile of xenograft rejection may resemble disease recurrence more that allograft rejection.
  • the immune system In the case of disease recurrence, the immune system has already been activated, as evidenced by destruction of the native islet cells. Therefore, in disease recurrence the immune system is already at the effector stage.
  • Antagonists of the present invention are able to suppress the immune response to both allografts and xenografts by decreasing the rate of TRl 1-, TRl 1SV1-, and TRl lSV2-mediated lymphocyte proliferation and differentiation.
  • Such antagonists include the TRl 1-, TRl 1SV1-, and TRl lSV2-Fc fusion proteins described in Example 5.
  • the present invention further provides a method for suppression of immune responses.
  • TNF-alpha has been shown to prevent diabetes in strains of animals which are prone to this affliction resulting from autoimmunity. See Porter, A., Tibtech 9:158-162 (1991).
  • agonists and antagonists of the present invention may be useful in the treatment, detection, and/or prevention of autoimmune diseases such as type 1 diabetes.
  • the role played by the TRl l, TRl lSVl, and TRHSV2 receptors in cell proliferation and differentiation indicates that agonist or antagonist of the present invention may be used to treat, detect, and/or prevent disease states involving aberrant cellular expression of these receptors.
  • TRl 1, TRl ISVl, and TRl 1SV2 receptors may in some circumstances induce an inflammatory response, and antagonists may be useful reagents for blocking this response.
  • TRl l, TRl lSVl, and TRHSV2 receptor antagonists e.g., soluble forms of the TRl 1, TRl ISVl, and TRl 1SV2 receptors; neutralizing antibodies
  • TRl l, TRl lSVl, and TRHSV2 receptor antagonists e.g., soluble forms of the TRl 1, TRl ISVl, and TRl 1SV2 receptors; neutralizing antibodies
  • TRl l, TRl lSVl, and TRHSV2 receptor antagonists may be useful for treating, detecting, and/or preventing inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
  • Antagonists to the TRl 1, TRl ISVl, and TRl 1SV2 receptor may also be employed to treat, detect, and or prevent and/or prevent septic shock, which remains a critical clinical condition.
  • Septic shock results from an exaggerated host response, mediated by protein factors such as TNF and EL-1, rather than from a pathogen directly.
  • protein factors such as TNF and EL-1
  • lipopolysaccharides have been shown to elicit the release of TNF leading to a strong and transient increase of its serum concentration.
  • TNF causes shock and tissue injury when administered in excessive amounts.
  • antagonists to the TRl l, TRllSVl, and TR11SV2 receptors will block the actions of TNF and treat, detect, and/or prevent septic shock.
  • These antagonists may also be employed to treat, detect, and/or prevent meningococcemia in children which correlates with high semm levels of TNF.
  • TRl l a ligand for TNF receptor ligands
  • TRl 1 a ligand for TNF receptor ligands
  • TRl 1 a ligand for TNF receptor ligands to TNF receptor ligands
  • TRl 1 a receptor for TNF receptor ligands to TNF receptor ligands to TNF, TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides
  • chemotaxis chemotaxis
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • the etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious.
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides can be used as a marker or detector of a particular immune system disease or disorder.
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may be useful in treating, detecting, and/or preventing deficiencies or disorders of hematopoietic cells.
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat, detect, and/or prevent those disorders associated with a decrease in certain (or many) types hematopoietic cells.
  • immunologic deficiency syndromes include, but are not limited to:blood protein disorders (e.g.
  • agammaglobulinemia agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
  • SIDs severe combined immunodeficiency
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides can also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity,
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides could be used to treat, detect, and or prevent blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
  • TRl 1 , TRl ISVl or TRl 1SV2 polynucleotides or polypeptides that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting, important in the treatment, detection, and/or prevention of heart attacks (infarction), strokes, or scarring.
  • TRl 1 , TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may also be useful in treatment, detection, and/or prevention of autoimmune disorders.
  • Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of TRll, TRllSVl or TRl 1SV2 polypeptides or polynucleotides that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
  • autoimmune disorders that can be treated, detected, and/or prevented by TRl 1, TRl ISVl or TRl 1SV2 include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • ERTAIN responses and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, detected, and/or prevented by TRl l, TRllSVl or TRl IS V2 polypeptides or polynucleotides.
  • TRl l, TRl ISVl or TRl 1SV2 can be used to treat, detect, and/or prevent anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may also be used to treat, detect, and/or prevent organ rejection or graft-versus-host disease (GVHD).
  • Organ rejection occurs by host immune cell destmction of the transplanted tissue through an immune response.
  • an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells may be an effective therapy in preventing organ rejection or GVHD.
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides may also be used to modulate inflammation.
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat, detect, and/or prevent inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or EL-1.)
  • infection e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury e.g., endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cyto
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides can be used to treat, detect, and/or prevent hyperproliferative disorders, including neoplasms.
  • TRll, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides may inhibit the proliferation of the disorder through direct or indirect interactions.
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides may proliferate other cells which can inhibit the hyperproliferative disorder.
  • hyperproliferative disorders can be treated, detected, and/or prevented.
  • This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • decreasing an immune response may also be a method of treating, detecting, and/or preventing hyperproliferative disorders, such as a chemotherapeutic agent.
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides examples include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
  • hyperproliferative disorders can also be treated, detected, and/or prevented by TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides.
  • hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • TRll, TRllSVl or TR11SV2 polypeptides or polynucleotides can be used to treat, detect, and/or prevent infectious agents.
  • infectious diseases may be treated, detected, and/or prevented.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • TRl 1, TRl ISVl or TRl 1SV2 polypeptides or polynucleotides may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • Vimses are one example of an infectious agent that can cause disease or symptoms that can be treated, detected, and or prevented by TR 11 , TR 11 S V 1 , TRl 1SV2 polynucleotides or polypeptides, or agonists of TRl 1, TRl ISVl, TRl 1SV2.
  • viruses include, but are not limited to the following DNA and RNA vimses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivims, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma vims, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (
  • Retroviridae HTLV-I, HTLV-II, Lentivims
  • Togaviridae e.g., Rubivims
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi
  • TRll, TRl lSVl, TR11SV2 polynucleotides or polypeptides, or agonists or antagonists of TRll, TRllSVl, TR11SV2, can be used to treat, detect, and/or prevent any of these symptoms or diseases.
  • TRl 1SV2 polynucleotides, polypeptides, or agonists are used to treat, detect, and/or prevent: meningitis, Dengue, EBV, and or hepatitis (e.g., hepatitis B).
  • TRl 1, TRl ISVl, TRl 1SV2 polynucleotides, polypeptides, or agonists are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines.
  • TRl 1, TRl ISVl, TR11SV2 polynucleotides, polypeptides, or agonists are used to treat, detect, and/or prevent AIDS.
  • TR 11 bacterial or fungal agents that can cause disease or symptoms and that can be treated, detected, and/or prevented by TR 11 , TR 11 S V 1 , TR 11 S V2 polynucleotides or polypeptides, or agonists or antagonists of TRll, TRllSVl,
  • TRl 1SV2 include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis, Candidiasis, Campylobacter,
  • Coccidioidomycosis Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxi genie E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickett
  • bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
  • TRll, TRllSVl, TR11SV2 polynucleotides or polypeptides, or agonists or antagonists of TRl 1, TRl ISVl, TRl 1SV2, can be used to treat, detect, and/or prevent any of these symptoms or diseases.
  • TRl 1, TRl ISVl, TRl 1SV2 polynucleotides, polypeptides, or agonists thereof are used to treat, detect, and/or prevent: tetanus, Diptheria, botulism, and/or meningitis type B.
  • parasitic agents causing disease or symptoms that can be treated by TRl l, TRl lSVl, TR11SV2 polynucleotides or polypeptides, or agonists of TRl l, TRl ISVl, TRl 1SV2, include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale).
  • TRl l, TRllSVl, TR11SV2 polynucleotides or polypeptides, or agonists or antagonists of TRl l, TRllSVl,
  • TRl 1SV2 can be used to treat or detect any of these symptoms or diseases.
  • TRl l, TRllSVl, TR11SV2 polynucleotides, polypeptides, or agonists thereof are used to treat malaria.
  • TRl 1, TRl ISVl, TRl 1SV2 polynucleotides or polypeptides, or agonists of TRl 1, TRllSVl, TR11SV2, is osteomyelitis.
  • treatment using TRl 1, TRllSVl, TR11SV2 polynucleotides or polypeptides, or agonists of TRll, TRllSVl, TR11SV2 could either be by administering an effective amount of TRl 1 , TRl ISVl, TRl 1SV2 polypeptide to the patient, or by removing cells from the patient, supplying the cells with TRl 1 ,
  • TRl ISVl TRl 1SV2 polynucleotide
  • returning the engineered cells to the patient (ex vivo therapy).
  • the TRl l, TRllSVl, TRl 1SV2 polypeptide or polynucleotide can be used as an adjuvant in a vaccine to raise an immune response against infectious disease.
  • TRll, TRl lSVl or TRl 1SV2 polynucleotides or polypeptides can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
  • the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, bu s, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs
  • TRl 1 , TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may increase regeneration of tissues difficult to heal. For example, increased tendon ligament regeneration would quicken recovery time after damage.
  • TRl l, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides of the present invention could also be used prophylactically in an effort to avoid damage.
  • Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.
  • tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by using TRl l, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke).
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. As a chemotactic molecule, TRl 1, TRl ISVl or TRl 1SV2 could also attract fibroblasts, which can be used to treat wounds.
  • TRll, TRllSVl or TR11SV2 polynucleotides or polypeptides may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, TRl 1, TRl ISVl or TRl 1SV2 polynucleotides or polypeptides could be used as an inhibitor of chemotaxis.
  • Additional preferred embodiments of the invention include, but are not limited to, the use of TRl l, TRllSVl, TR11SV2 polypeptides and functional agonists in the following applications:
  • an animal e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human
  • boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
  • an animal including, but not limited to, those listed above, and also including transgenic animals
  • an animal incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741.
  • a vaccine adjuvant that enhances immune responsiveness to specific antigen.
  • compositions of the invention include virus and virus associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B).
  • compositions of the invention are used as an adjuvant to enhance an immune response to a vims, disease, or symptom selected from the group consisting of: HJV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovims, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever.
  • a vims, disease, or symptom selected from the group consisting of: HJV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovims, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever.
  • compositions of the invention include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
  • compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).
  • compositions of the invention include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria).
  • compositions of the invention may be administered prior to, concomitant with, and/or after transplantation.
  • compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations.
  • compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
  • B cell immunodeficiencies that may be ameliorated or treated by administering the TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, SCID, congenital agammaglobulinemia, common variable immunodeficiency, Wiskott-Aldrich Syndrome, X-linked immunodeficiency with hyper IgM, and severe combined immunodeficiency.
  • Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, HJV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
  • CLL B cell chronic lymphocytic leukemia
  • Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.
  • viral infections e.g., influenza
  • TRl 1, TRl ISVl, TRl 1SV2 enhances antigen presentation or antagonizes antigen presentation in vitro or in vivo.
  • said enhancement or antagonization of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
  • a humoral response i.e. TH2
  • TH1 humoral response
  • multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
  • TRl ISVl As an antigen for the generation of antibodies to inhibit or enhance TRl 1, TRl ISVl, TRl 1SV2 mediated responses. As a means of activating T cells.
  • monocytes/macrophages As a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leshmania.
  • TRl lSVl TRl 1SV2.
  • TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or agonists may be used to modulate IgE concentrations in vitro or in vivo.
  • TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or agonists thereof may be used to treat or prevent IgE-mediated allergic reactions.
  • allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.
  • Antagonists of TR 11 , TR 11 S V 1 , TR 11 S V2 include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the TRl l, TRl ISVl, TRl 1SV2 receptor(s) (e.g., a TRl 1-Fc fusion protein containing amino acids of SEQ ID NO:2) (see e.g., Example 31). These would be expected to reverse many of the activities of the ligand described above as well as find clinical or practical application as:
  • TRl 1, TRl ISVl, TRl 1SV2 may, like CD40 and its ligand, be regulated by the status of the immune system and the microenvironment in which the cell is located.
  • a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythramatosus and MS.
  • An inhibitor of B and or T cell migration in endothelial cells This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
  • a therapy for B cell and/or T cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.
  • MGUS monoclonalgammopathy of undetermined significance
  • Waldenstrom's disease Waldenstrom's disease
  • related idiopathic monoclonalgammopathies and plasmacytomas.
  • An immunosuppressive agent(s) is an immunosuppressive agent(s).
  • TRl 1, TRl ISVl, TRl 1SV2 polypeptides or polynucleotides of the invention, or antagonists may be used to modulate IgE concentrations in vitro or in vivo.
  • administration of TRl l , TRl lSVl, TR11SV2 polypeptides or polynucleotides of the invention, or antagonists thereof may be used to treat or prevent IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis, and eczema.
  • IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis, and eczema.
  • An inhibitor of signaling pathways involving ERK1, COX2 and Cyclin D2 which have been associated with TRl 1, TRl ISVl and/or TRl 1SV2 induced B cell activation.
  • the agonists and antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described above.
  • the antagonists may be employed for instance to inhibit TRl 1, TRl ISVl and/or TRl 1SV2 chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain auto-immune and chronic inflammatory and infective diseases.
  • auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes.
  • the antagonists may also be employed to treat infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the recmitment and activation of mononuclear phagocytes. They may also be employed to treat idiopathic hyper-eosinophilic syndrome by preventing eosinophil production and migration. Endotoxic shock may also be treated by the antagonists by preventing the migration of macrophages and their production of the TRl 1, TRl ISVl and/or TRl 1SV2 polypeptides of the present invention. The antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall.
  • the antagonists may also be employed to treat histamine-mediated allergic reactions and immunological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and basophil degranulation and release of histamine.
  • IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema may also be treated.
  • the antagonists may also be employed to treat chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung.
  • Antagonists may also be employed to treat rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies.
  • the antagonists may be employed to interfere with the deleterious cascades attributed primarily to IL-1 and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be employed to prevent inflammation.
  • the antagonists may also be employed to inhibit prostaglandin-independent fever induced by TRl 1, TRl ISVl and/or TRl 1SV2.
  • the antagonists may also be employed to treat cases of bone marrow failure, for example, aplastic anemia and myelodysplastic syndrome.
  • the antagonists may also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung.
  • the antagonists may also be employed to treat subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung.
  • Antibodies against TRl 1, TRl ISVl and/or TRl 1SV2 may be employed to bind to and inhibit TRl 1, TRl ISVl and/or TRl 1SV2 activity to treat ARDS, by preventing infiltration of neutrophils into the lung after injury.
  • the antagonists and antagonists of the instant may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described hereinafter.
  • Agonists and antagonist of the invention also have uses in stimulating wound and tissue repair, stimulating angiogenesis, stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
  • Polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
  • diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g., HJV-1 infection, HJN-2 infection, herpesvirus infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovi
  • osteomyelodysplasia e.g., aplastic anemia, etc.
  • liver disease e.g., acute and chronic hepatitis, liver injury, and cirrhosis
  • autoimmune disease e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.
  • cardiomyopathy e.g., dilated cardiomyopathy
  • diabetes diabetic complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, regulating hematopoiesis, wound healing (e.g., wounds, bums, and bone fractures).
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen and/or anti-viral immune responses. More generally, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response. For example, polynucleotides and/or polypeptides of the invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
  • polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders.
  • polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
  • hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human.
  • the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat.
  • the host is a mammal.
  • the host is a human.
  • the agonist or antagonists described herein can be administered in vitro, ex vivo, or in vivo to cells which express the receptor of the present invention.
  • administration of an "effective amount" of an agonist or antagonist is intended an amount of the compound that is sufficient to enhance or inhibit a cellular response to a TNF-family ligand and include polypeptides.
  • administration of an "effective amount” of an agonist or antagonists is intended an amount effective to enhance or inhibit TRl 1 , TRl 1 SV1 , and TRl 1SV2 receptor mediated activity.
  • an agonist according to the present invention can be co-administered with a TNF-family ligand.
  • an agonist or antagonist can be determined empirically and may be employed in pure form or in pharmaceutically acceptable salt, ester or pro-dmg form.
  • the agonist or antagonist may be administered in compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon factors well known in the medical arts.
  • the total pharmaceutically effective amount of a TRl 1, TRl ISVl or TRl 1SV2 polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
  • the TRll, TRllSVl, and TRl 1SV2 polypeptides are typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump.
  • An intravenous bag solution may also be employed.
  • compositions containing the TRl 1, TRl ISVl, and TRl 1SV2 receptor polypeptides of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.
  • compositions of the invention may be administered alone or in combination with other adjuvants.
  • Adjuvants that may be administered with the compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL.
  • compositions of the invention are admninistered in combination with alum.
  • compositions of the invention are administered in combination with QS-21.
  • compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.
  • Vaccines that may be administered with the compositions of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavims, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially.
  • Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • compositions of the invention may be administered alone or in combination with other therapeutic agents.
  • Therapeutic agents that may be administered in combination with the compositions of the invention include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors.
  • Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual.
  • 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 with the compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-lBBL, DcR3, OX40L, TNF-gamma (International Publication No.
  • WO 96/14328 AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No.
  • compositions of the invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g.,
  • AVRENDTM bioloigically active fragments, variants, or derivatives of CD40L, anti-
  • CD40L antibodies e.g,. agonistic or antagonistic antibodies
  • anti-CD40 antibodies e.g, agonistic or antagonistic antibodies
  • 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 transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, RETRO VIRTM
  • Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and
  • Protease inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, CRIXIVANTM
  • antiretroviral agents nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat
  • compositions of the invention may be administered in combination with anti-opportunistic infection agents.
  • Anti-opportunistic agents that may be administered in combination with the compositions of the invention, include, but are not limited to, TRIMETHOPPJM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMEDINETM, ATOVAQUONETM, ISONIAZIDTM, RTFAMPINTM, PYRAZINAMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMENETM, LEUCOVORINTM, NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM (sargramostim/GM-CSF
  • compositions of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and or ATOVAQUONETM to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection.
  • compositions of the invention are used in any combination with ISONIAZEDTM,
  • compositions of the invention are used in any combination with REFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection.
  • compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat or prevent an opportunistic cytomegalovirus infection.
  • compositions of the invention are used in any combination with
  • compositions of the invention are used in any combination with
  • compositions of the invention are used in any combination with
  • compositions of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat or prevent an opportunistic bacterial infection.
  • compositions of the invention are administered in combination with an antiviral agent.
  • Antiviral agents that may be administered with the compositions of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
  • the compositions of the invention are administered in combination with an antibiotic agent.
  • Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
  • Nonspecific immunosuppressive agents that may be administered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
  • immunosuppressants preparations that may be administered with the compositions of the invention include, but are not limited to, ORTHOCLONETM (OKT3), SANDIMMUNETM/NEORALTM/SANGDYATM (cyclosporin), PROGRAFTM (tacrolimus), CELLCEPTTM (mycophenolate), Azathioprine, glucorticosteroids, and
  • compositions of the invention are administered in combination with steroid therapy.
  • Steroids that may be administered in combination with the compositions of the invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone).
  • compositions of the invention are administered in combination with prednisone.
  • the compositions of the invention are administered in combination with prednisone and an immunosuppressive agent.
  • Immunosuppressive agents that may be administered with the compositions of the invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
  • compositions of the invention are administered in combination with methylprednisolone.
  • the compositions of the invention are administered in combination with methylprednisolone and an immunosuppressive agent.
  • Immunosuppressive agents that may be administered with the compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
  • compositions of the invention are administered in combination with an antimalarial.
  • Antimalarials that may be administered with the compositions of the invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.
  • compositions of the invention are administered in combination with an NSAED.
  • compositions of the invention are administered in combination with one, two, three, four, five, ten, or more of the following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), kornac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-lRa gene therapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone me
  • NRD-101 Ho
  • compositions of the invention are administered in combination with one, two, three, four, five or more of the following dmgs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial dmg (e.g., as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti-TNF antibody, and prednisolone.
  • compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBRELTM and/or suflasalazine.
  • the compositions of the invention are administered in combination with methotrexate.
  • the compositions of the invention are administered in combination with anti-TNF antibody.
  • the compositions of the invention are administered in combination with methotrexate and anti-TNF antibody.
  • the compositions of the invention are administered in combination with suflasalazine.
  • the compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine.
  • compositions of the invention are administered in combination with ENBRELTM and methotrexate. In another embodiment, the compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one of the above-recited combinations. In a specfic embodiment, the compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELTM, methotrexate and suflasalazine.
  • an antimalarial e.g., hydroxychloroquine
  • compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and methotrexate.
  • compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.
  • Intravenous immune globulin preparations that may be administered with the compositions of the invention include, but not limited to, GAMMARTM, IVEEGAMTM,
  • compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
  • transplantation therapy e.g., bone marrow transplant.
  • CD40 ligand CD40L
  • a soluble form of CD40L e.g., AVRENDTM
  • biologically active fragments, variants, or derivatives of CD40L e.g., anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
  • compositions of the invention are administered alone or in combination with an anti-inflammatory agent.
  • Anti-inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4- hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline
  • compositions of the invention are administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that may be administered with the compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxombicin, bleomycin, daunombicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha- 2b, glutamic acid, plicamycin, mercaptopurine, and 6- thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,
  • compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxombicin, vincristine, and prednisone) or any combination of the components of CHOP.
  • CHOP cyclophosphamide, doxombicin, vincristine, and prednisone
  • compositions of the invention are administered in combination with Rituximab.
  • compositions of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.
  • the compositions of the invention are administered in combination with cytokines.
  • Cytokines that may be administered with the compositions of the invention include, but are not limited to, GM-CSF, G-CSF, IL- lalpha, IL-lbeta, 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, anti-CD40, CD40L, IFN-alpha, EFN-beta, IFN-gamma, TNF-alpha, and TNF-beta.
  • compositions of the invention are administered in combination with angiogenic proteins.
  • Angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to,. Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP- 682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (P1GF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477;
  • compositions of the invention are administered in combination with Fibroblast Growth Factors.
  • Fibroblast Growth Factors tha may be administered with the compositions of the 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.
  • compositions of the invention are administered in combination with hematopoietic growth factors.
  • Hematopoietic growth factors that may be administered with the compositions of the invention included, but are not limited to, LEUKINETM (SARGRAMOSTIMTM) and NEUPOGENTM
  • compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • Example 1 Expression and Purification of TRll in E. coli.
  • the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311).
  • pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo- tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN, Inc., supra, and suitable single restriction enzyme cleavage sites.
  • a DNA fragment encoding a polypeptide may be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
  • the polypeptide coding sequence is inserted such that translation of the six His codons is prevented and, therefore, the polypeptide is produced with no 6 X His tag.
  • the novel ⁇ HE4 series of bacterial expression vectors in particular, the pHE4-5 vector may be used for bacterial expression in this example.
  • the pHE4-5/MPIFD23 vector plasmid DNA containing an insert which encodes another ORF (using the NJe I and Asp 718 flanking restriction sites, one of ordinary skill in the art could easily use current molecular biological techniques to replace the irrelevent ORF in the pHE4-5 vector with the ORF of the present invention) was deposited on September 30, 1997 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and given ATCC Deposit No. 209311.
  • the bacterial expression vector pHE4-5 includes a neomycin phosphotranferase gene for selection, an E. coli origin of replication, a T5 phage promoter sequence, two lac operator sequences, a Shine-Delgarno sequence, and the lactose operon repressor gene (laclq).
  • the promoter and operator sequences of the pHE4-5 vector were made synthetically. Synthetic production of nucleic acid sequences is well known in the art (CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303).
  • the DNA sequence encoding the desired portion of the TRl 1 protein lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino terminal sequences of the desired portion of the TRl 1 protein and to sequences in the deposited constmct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
  • the 5' primer has the sequence: 5'-CGC CCA TGG CAG CGC CCC ACC G-3' (SEQ ID NO: 10) containing the underlined Nco I restriction site followed by 13 nucleotides complementary to the amino terminal coding sequence of the extracellular domain of the TRl 1 sequence in Figures 1A and IB (nucleotides 184-195 of SEQ ID NO:l).
  • SEQ ID NO: 10 amino terminal coding sequence of the extracellular domain of the TRl 1 sequence in Figures 1A and IB (nucleotides 184-195 of SEQ ID NO:l).
  • the point in the protein coding sequence where the 5' primer begins may be varied to amplify a desired portion of the complete protein shorter or longer than the mature form.
  • the 3' primer for the souble extracellular domain has the sequence: 5' CGC AAG CTT GGC TCT GCC GGC G 3' (SEQ ID NO: 11) containing the underlined Hindlll restriction site followed by 13 nucleotides complementary to the 3' end of the extracellular domain portion of the nucleotide sequence shown in Figures 1A and IB (nucleotides 590-602 in SEQ ID NO: 1) encoding the extracellular domain of the TRl 1 receptor.
  • the amplified TRl 1 DNA fragments and the vector pQE60 are digested with
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing TRl 1 protein, is available commercially from QIAGEN, Inc., supra.
  • Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constmcts are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor.
  • Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.
  • the cells are then stirred for 3-4 hours at 4°C in 6 M guanidine-HCl, pH 8.
  • the cell debris is removed by centrifugation, and the supernatant containing the TRl 1 extracellular domain polypeptide is dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with 200 mM NaCl.
  • the protein can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4, containing protease inhibitors.
  • the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography.
  • an affinity chromatography step such as an antibody column can be used to obtain pure TRl 1 extracellular domain polypeptide.
  • the purified protein is stored at 4°C or frozen at -80°C.
  • the plasmid shuttle vector pA2GP was used to insert the cloned
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis vims (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as Bam HI, Xba I and Asp 718.
  • the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable vims that expresses the cloned polynucleotide.
  • baculovims vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the constmct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
  • Such vectors are described, for instance, in Luckow et al, Virology 170:31- 39.
  • the cDNA sequence encoding essentially the extracellular domain with leader (amino acids 1 to 162 shown in Figures 1A and IB) of the TRl 1 receptor protein in the deposited clone (ATCC Deposit Number 209341) is amplified using PCR oligonucleotide primers corresponding to the relevant 5' and 3' sequences of the gene.
  • the 5' primer for the above has the sequence: 5-CGC GGA TCC CAG CGC CCC ACC G-3' (SEQ ED NO: 12) containing the underlined Bam HJ restriction enzyme site, an efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J Mol. Biol.
  • the 3' primer has the sequence: 5' CGC GGT ACC GGC TCT GCC GGC G-3' (SEQ ID NO:13) containing the underlined Asp 718 restriction sites followed by 13 nucleotides complementary to the coding sequence in Figures 1A and IB (nucleotides 590-602 in SEQ ID NO: 1).
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with Bam HI and Asp 718 and purified on a 1% agarose gel. This fragment is designated herein "FI".
  • the plasmid is digested with the restriction enzymes Bam Ffl and Asp 718 dephosphorylated using calf intestinal phosphatase.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, CA). This vector DNA is designated herein "VI”.
  • E. coli HB101 cells are transformed with the ligation mixture and spread on culture plates.
  • Other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) may also be used.
  • Bacteria are identified that contain the plasmid with the human TRl 1 sequences using the PCR method, in which one of the above primers is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing TRl 1 gene fragments show amplification of the DNA. The sequence of the cloned fragment is confirmed by DNA sequencing.
  • the plasmid is designated herein pBacTRl 1-T.
  • pBacTRl 1-T Five micrograms of pBacTRl 1-T is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovims DNA ("BaculoGold baculovims DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-1417 (1987). 1 ⁇ g of BaculoGold virus DNA and 5 ⁇ g of plasmid pBacTRl 1-T are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra.
  • An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques.
  • a detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf).
  • the agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ⁇ l of Grace's medium and the suspension containing the recombinant baculovims is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C.
  • the recombinant virus is called V-TR11-T.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat inactivated FBS. The cells are infected with the recombinant baculovims V-TR11-T at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD). Forty-two hours later, 5 ⁇ Ci of 35s-methionine and 5 ⁇ Ci 35s-cysteine (available from Amersham) are added to radiolabel proteins. The cells are further incubated for 16 hours and then they are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography. Microsequencing of the amino acid sequence of the amino terminus of purified protein is used to determine the amino terminal sequence of the mature protein and thus the cleavage point and length of the secretory signal peptide.
  • Example 2(b) Cloning and Expression of the Full-Length Gene for TRll Protein in a Baculovirus Expression System Similarly to the cloning and expression of the tmncated version of the TRl 1 receptor described in Example 2(a), recombinant baculovimses were generated which express the full length TRl 1 receptor protein shown in Figures 1 A and IB (SEQ ID NO:2).
  • the plasmid shuttle vector pA2 is used to insert the cloned DNA encoding the complete protein, including its naturally associated secretary signal (leader) sequence, into a baculovirus to express the mature TRl 1 protein.
  • leader naturally associated secretary signal
  • Other attributes of the pA2 vector are as described for the pA2GP vector used in Example 2(a).
  • the 5' primer for the above has the sequence: 5-CGC GGA TCC CCG CCA TCk TGG CAC AGC ACG GGG CG-3' (SEQ ID NO: 14) containing the underlined Bam Ffl restriction enzyme site, an efficient signal for initiation of translation in eukaryotic cells (in italics), as described by Kozak, M., J. Mol. Biol.
  • a suitable 3' primer for this purpose has the sequence: 5' CGC GGT ACC CAC CCA CAG GTC TCC C-3' (SEQ ID NO: 15) containing the underlined Asp 718 restriction sites followed by 16 nucleotides complementary to the coding sequence in Figures 1A and IB (nucleotides 804-819 in SEQ ED NO:l).
  • the amplified fragment is isolated and digested with restriction enzymes as described in Example 2(a) to produce plasmid pBacTRl 1
  • pBacTRl l 5 ⁇ g of pBacTRl l is co-transfected with 1 ⁇ g of BaculoGold (Pharmingen) viral DNA and 10 ⁇ l of Lipofectin (Life Technologies, Inc.) in a total volume of 200 ⁇ l semm free media.
  • the primary vimses are harvested at 4-5 days post-infection (pi), and used in plaque assays. Plaque purified vimses are subsequently amplified and frozen, as described in Example 2(a).
  • Sf9 cells are seeded in 12 well dishes with 2.0 ml of a cell suspension containing 0.5 x 10 ⁇ cells/ml and allowed to attach for 4 hours.
  • Recombinant'baculoviruses are used to infect the cells at an MOI of 1-2.
  • the media is replaced with 1.0 ml of serum free media depleted for methionine and cysteine (-Met -Cys).
  • the culture media is replaced with
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retrovimses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovims (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
  • LTRS long terminal repeats
  • Retrovimses e.g., RSV, HTLVI, HIVI
  • CMV cytomegalovims
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that could be used include, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded protein.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS; Murphy et al, Biochem J. 227:277-279 (1991); Bebbington et al, Bio/Technology 70:169-175 (1992)).
  • the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
  • These cell lines contain the amplified gene(s) integrated into a chromosome.
  • Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 47:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites Bam TTT, Xba I and Asp 718, facilitate the cloning of the gene of interest.
  • the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, pTRl 1HA is made by cloning a cDNA encoding the soluble extracellular portion of the TRl 1 protein into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • HA hemagglutinin fragment
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37:767 (1984).
  • the fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope.
  • pcDNAIII contains, in addition, the selectable neomycin marker.
  • a DNA fragment encoding a TRl 1 protein is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the plasmid constmction strategy is as follows.
  • the TRl 1 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of TRl 1 in E. coli.
  • Suitable primers include the following, which are used in this example.
  • the 5' primer containing the underlined Bam HI site, a Kozak sequence (in italics), an AUG start codon and 13 additional codons of the 5' coding region of the complete TRl 1 has the following sequence: 5'-CGC GGA TCC GCC ATC ATG CAG CGC CCC ACC G-3 * (S ⁇ Q ID NO: 16).
  • the 3' primer has the sequence: 5' CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA TTA GGC TCT GCC GGC G-3' (SEQ ID NO: 17) containing the underlined Xba I restriction site followed by a stop codon, a sequence encoding a 6x his tag, and 15 nucleotides complementary to the coding sequence in Figures 1A and IB (nucleotides 590-602 in SEQ ID NO:l).
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Bam TTT and Xba I and then ligated. The ligation mixture is transformed into E.
  • Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the TRl 1 -encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of TRl 1 by the vector.
  • TRl 1-HA fusion protein Expression of the TRl 1-HA fusion protein is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow et al, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the
  • 35 cells are labeled by incubation in media containing [ S]-cysteine for 8 hours.
  • the cells and the media are collected, and the cells are washed and lysed with detergent- containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Plasmid pC4 is used for the expression of TRl 1 protein.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • the amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Vims (Cullen, et al, Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovims (CMV) (Boshart et al, Cell 47:521-530 (1985)). Downstream of the promoter are Bam HI, Xba I, and Asp 718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • LTR long terminal repeat
  • CMV cytomegalovims
  • high efficiency promoters can also be used for the expression, e.g., the human beta-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the TRll protein in a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pC4 is digested with the restriction enzymes Bam HI and Asp 718 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete TRl 1 protein including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene having, for instance, the same sequences as the 5' and 3' primers used for cloning in baculovims pA vectors as shown in Example 2, above.
  • the amplified fragment is digested with the endonucleases Bam HI and Asp 718 and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli YTBlOl or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary cells lacking an active DHFR gene are used for transfection.
  • 5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin (Feigner et al, supra).
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.

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Abstract

L'invention concerne des éléments nouveaux de la famille des récepteurs TNF. Elle concerne des molécules d'acides nucléiques isolées codant les récepteurs humains TR11, TR11SV1, et TRSV2. L'invention concerne également des polypeptides TR11, TR11SV1, et TR11SV2, de même que des vecteurs, des cellules hôtes et des procédés de recombinaison qui permettent leur élaboration. L'invention concerne en outre des procédés de criblage permettant d'identifier des agonistes et des antagonistes vis-à-vis de l'activité des récepteurs TR11, TR11SV1, et TR11SV2. L'invention concerne par ailleurs des procédés diagnostiques permettant de déceler les états pathologiques liés à l'expression aberrante des récepteurs TR11, TR11SV1, et TR11SV2. L'invention concerne enfin des procédés thérapeutiques permettant de traiter les états pathologiques liés à la prolifération et à la différenciation aberrantes des cellules qui expriment les récepteurs TR11, TR11SV1, et TR11SV2.
PCT/US2000/004572 1999-02-24 2000-02-23 Proteines tr11, tr11sv1, et tr11sv2 du type recepteur tnf humain WO2000050459A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003049756A1 (fr) * 2001-12-13 2003-06-19 Genset S.A. Agonistes et antagonistes de glucomine a utiliser dans le traitement de troubles metaboliques
WO2003051386A1 (fr) * 2001-12-14 2003-06-26 Genset S.A. Agonistes et antagonistes de glucoset a utiliser dans le traitement de troubles metaboliques
WO2006078911A3 (fr) * 2005-01-19 2007-06-28 Genzyme Corp Anticorps anti-gitr pour le diagnostic du cancer du poumon non a petites cellules (cpnpc)
US9228016B2 (en) 2014-06-06 2016-01-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US9701751B2 (en) 2009-09-03 2017-07-11 Merck Sharp & Dohme Corp. Anti-GITR antibodies
US11213586B2 (en) 2015-11-19 2022-01-04 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR)
US11408889B2 (en) 2015-06-03 2022-08-09 Bristol-Myers Squibb Company Anti-GITR antibodies for cancer diagnostics
US11685787B2 (en) 2017-05-16 2023-06-27 Bristol-Myers Squibb Company Treatment of cancer with anti-GITR agonist antibodies

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009386A2 (fr) * 1994-09-21 1996-03-28 The Government Of The United States Of America, Re Variantes alleliques du recepteur 5ht2c de la serotonine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009386A2 (fr) * 1994-09-21 1996-03-28 The Government Of The United States Of America, Re Variantes alleliques du recepteur 5ht2c de la serotonine

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003049756A1 (fr) * 2001-12-13 2003-06-19 Genset S.A. Agonistes et antagonistes de glucomine a utiliser dans le traitement de troubles metaboliques
WO2003051386A1 (fr) * 2001-12-14 2003-06-26 Genset S.A. Agonistes et antagonistes de glucoset a utiliser dans le traitement de troubles metaboliques
WO2006078911A3 (fr) * 2005-01-19 2007-06-28 Genzyme Corp Anticorps anti-gitr pour le diagnostic du cancer du poumon non a petites cellules (cpnpc)
US10400040B2 (en) 2009-09-03 2019-09-03 Merck Sharp & Dohme Corp. Anti-GITR antibodies
US9701751B2 (en) 2009-09-03 2017-07-11 Merck Sharp & Dohme Corp. Anti-GITR antibodies
US9228016B2 (en) 2014-06-06 2016-01-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US9745379B2 (en) 2014-06-06 2017-08-29 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US10465010B2 (en) 2014-06-06 2019-11-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US10501550B2 (en) 2014-06-06 2019-12-10 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11084881B2 (en) 2014-06-06 2021-08-10 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11802162B2 (en) 2014-06-06 2023-10-31 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11408889B2 (en) 2015-06-03 2022-08-09 Bristol-Myers Squibb Company Anti-GITR antibodies for cancer diagnostics
US11213586B2 (en) 2015-11-19 2022-01-04 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR)
US11685787B2 (en) 2017-05-16 2023-06-27 Bristol-Myers Squibb Company Treatment of cancer with anti-GITR agonist antibodies

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