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WO2000047740A2 - Proteines associees au facteur de necrose des tumeurs - Google Patents

Proteines associees au facteur de necrose des tumeurs Download PDF

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WO2000047740A2
WO2000047740A2 PCT/US2000/003653 US0003653W WO0047740A2 WO 2000047740 A2 WO2000047740 A2 WO 2000047740A2 US 0003653 W US0003653 W US 0003653W WO 0047740 A2 WO0047740 A2 WO 0047740A2
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agp
seq
polypeptide
protein
cell
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PCT/US2000/003653
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WO2000047740A3 (fr
WO2000047740A9 (fr
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William J. Boyle
Hailing Hsu
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Amgen Inc.
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Publication of WO2000047740A3 publication Critical patent/WO2000047740A3/fr
Publication of WO2000047740A9 publication Critical patent/WO2000047740A9/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
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates to proteins that are involved in inflammation and immunomodulation, particularly in B cell growth, survival, or activation.
  • the invention further relates to proteins related to the tumor necrosis factor (TNF) /nerve growth factor (NGF) superfamily and related nucleic acids, expression vectors, host cells, and binding assays.
  • TNF tumor necrosis factor
  • NGF nerve growth factor
  • the specification also describes compositions and methods for the treatment of immune-related and inflammatory, autoimmune and other immune-related diseases or disorders, such as rheumatoid arthritis (RA), Crohn's disease (CD), lupus, and graft versus host disease (GvHD).
  • RA rheumatoid arthritis
  • CD Crohn's disease
  • GvHD graft versus host disease
  • the invention also relates to methods and compositions for the treatment of inflammatory and immune-related diseases and disorders using the receptors.
  • TNFs tumor necrosis factors
  • CD40L TNF-related apoptosis-inducing ligand
  • TRAIL TNF-related apoptosis-inducing ligand
  • AGP-1 TNF-related apoptosis-inducing ligand
  • OPG-BP or OPG ligand osteoprotegerin binding protein
  • 4-1BB ligand LIGHT, APRIL, and TALL-1.
  • Leukocyte Biology 65: 680-3 This family is unified by its structure, particularly at the C- terrriinus. In addition, most members known to date are expressed in immune compartments, although some members are also expressed in other tissues or organs, as well. Smith et al. (1994), Cell 76: 959-62. All ligand members, with the exception of LT- ⁇ , are type II transmembrane proteins, characterized by a conserved 150 amino acid region within the C-terminal extracellular domain. Though restricted to only 20-25% identity, the conserved 150 amino acid domain folds into a characteristic ⁇ -pleated sheet sandwich and trimerizes. This conserved region can be proteolyticaly released, thus generating a soluble functional form. Banner et al. (1993), Cell 73: 431 ⁇ 45.
  • TNF ⁇ is mainly synthesized by macrophages and is an important mediator for inflammatory responses and immune defenses.
  • Fas-L predominantly expressed in activated T cell, modulates TCR-mediated apoptosis of thymocyts. Nagata, S. & Suda, T. (1995) Immunology Today 16: 39-43; Castrim et al. (1996), Immunity 5: 617-27.
  • CD40L also expressed by activated T cells, provides an essential signal for B cell survival, proliferation and immunoglobulin isotype switching. Noelle (1996), Immunity 4: 415-9.
  • the cognate receptors for most of the TNF ligand family members have been identified. These receptors share characteristic multiple cysteine-rich repeats within their extracellular domains, and do not possess catalytic motifs within cytoplasmic regions. Smith et al. (1994).
  • the receptors signal through direct interactions with death domain proteins (e.g. TRADD, FADD, and RIP) or with the TRAF proteins (e.g. TRAF2, TRAF3, TRAF5, and TRAF6), triggering divergent and overlapping signaling pathways, e.g. apoptosis, NF- ⁇ B activation, or JNK activation. Wallach et al. (1999), Annual Review of Immunology 17: 331- 67. These signaling events lead to cell death, proliferation, activation or differentiation.
  • each receptor member varies.
  • TNFR1 is expressed on a broad spectrum of tissues and cells; whereas the cell surface receptor of OPGL is mainly restricted to the osteoclasts. Hsu et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3540-5. It is therefore an object of the invention to identify proteins and nucleic acids related to TNFs. Such proteins are believed to play a role in inflammatory and immune processes, suggesting their usefulness in treating autoimmune and inflammatory disorders.
  • the novel TNF ligand family member is herein called AGP-3.
  • AGP-3 The novel TNF ligand family member is primarily expressed in B cells, and its expression correlates to increases in the number of B cells and immunoglobulins produced.
  • the natural, preferred human ortholog is here called hAGP-3 and contains 285 amino acids; the mouse ortholog (mAGP-3), contains 309 amino acids.
  • the AGP-3 protein is a type II transmembrane protein and has an amino terrninal cytoplasmic domain, a transmembrane domain, and a carboxy terminal extracellular domain.
  • TNF-related proteins of the invention may be membrane-associated or in soluble form, recombinantly produced or isolated after natural production.
  • AGP-3 is a potent B cell stimulatory factor.
  • the AGP-3 transgenic mice also developed autoantibodies and kidney immune complex deposits, a phenotype resembling lupus patients and lupus prone mice.
  • the invention provides for nucleic acids encoding AGP-3, vectors and host cells expressing the polypeptides, and methods for producing recombinant proteins. Antibodies or fragments thereof that specifically bind AGP-3 are also provided.
  • the subject proteins may be used in assays to identify cells and tissues that express AGP-3 or proteins related to AGP-3 and to identify new AGP-3-related proteins.
  • Methods of identifying compounds that interact with AGP-3 proteins are also provided.
  • Such compounds include nucleic acids, peptides, proteins, carbohydrates, lipids or small molecular weight organic molecules and may act either as agonists or antagonists of AGP-3 or AGP-3 R-protein activity.
  • AGP-3-related proteins are involved in B cell growth, survival, and activation, particularly in lymph node, spleen, and Peyer's patches.
  • AGP-3 agonists and antagonists e.g., antibodies to AGP-3 thus modulate B cell response and may be used to treat diseases characterized by inflarr-matory processes or deregulated immune response, such as RA, GvHD, CD, lupus, and the like.
  • Pharmaceutical compositions comprising AGP-3- related proteins and AGP-3 agonists and antagonists are also encompassed by the invention.
  • AGP-3 related proteins may also be useful in production of hybridoma cells, which are derived from B cells.
  • the present invention also concerns a method to modulate hybridoma cell antibody production, which comprises treating hybridoma cells with the compound of Claim 1.
  • Figure 1 shows the sequence of human AGP-3. Nucleic acid and amino acid sequences of human AGP-3 are indicated (SEQ ID NOS: 1 and 2, respectively). The predicted transmembrane region is underlined. Potential N-linked glycosylation sites are shown in boldface.
  • Figure 2 shows the sequence of murine AGP-3. Nucleic acid and amino acid sequences of murine AGP-3 are indicated (SEQ ID NOS: 3 and 4, respectively). The predicted transmembrane region is underlined. Potential N-linked glycosylation sites are shown in boldface.
  • Figure 3 shows an alignment of human and murine AGP-3, along with a consensus sequence (SEQ ID NO: 5).
  • the predicted human and murine AGP-3 protein sequences were aligned by Pileup with gap creation penalty (12) and gap extension penalty (4) (Wisconsin GCG Package, Version 8.1, Genetics Computer Group Inc., Madison, Wisconsin).
  • the consensus sequence was determined by Lineup (Wisconsin GCG Package, Version 8.1).
  • the transmembrane regions from amino acid 47 to 72 in human AGP-3 and from amino acid 48 to 73 in murine AGP-3 are underlined.
  • the N-te ⁇ ninal intracellular domain resides from amino acid 1 to 46 in human AGP-3 and from amino acid 1 to 47 in murine AGP-3.
  • the C-terminal extracellular domain is localized from amino acid 73 to 285 in human AGP-3, and from amino acid 74 to 309.
  • the human and murine AGP-3 share 68% arnino acid identity overall.
  • the C-terminus of AGP-3 is more conserved between human and mouse, with 87% identity over a 142-amino acid length.
  • the putative conserved beta strands are indicated at the top, with the amino acids forming the putative strands underlined.
  • FIG 4 shows human and murine AGP-3 mRNA tissue distribution.
  • Human tissue northern blots (A) and murine tissue northern blots (B) were probed with 32 P-labeled human AGP-3 probe (A) or murine AGP-3 probe. The probed blots were exposed to Kodak film for 18 hours (A) or seven days (B).
  • Figure 5 shows histology analysis of AGP-3 transgenic mouse spleen. The spleen sections from control mouse (A, C and E) and AGP-3 transgenic mouse (B, D, and F) were stained with hematoxylin and exosin (A and B), anti-mouse B220 (C and D) or anti-mouse CD3 (E and F).
  • the spleen of the transgenic mouse was enlarged, mainly due to the increase of size and number of the follicles.
  • the B cell staining areas in the spleen follicles in the transgenic mouse were enlarged.
  • the T cell number was slightly diminished.
  • FIG. 6 shows histology analysis of AGP-3 transgenic mouse lymph nodes.
  • the lymph node sections from control mouse (A, C and E) and AGP-3 transgenic mouse (B, D, and F) were stained with hematoxylin and exosin (A and B), anti-mouse B220 (C and D) or anti-mouse CD3 (E and F).
  • the lymph node size of the transgenic mouse was enlarged.
  • the B cell number was greatly increased in the transgenic mouse. Instead of restricted to marginal zones of the follicles as in the control mouse, the B cells also filled out the follicular area in the lymph nodes of the transgenic mouse.
  • the T cell number was decreased in the transgenic mouse as compared to the control.
  • FIG 7 shows histology analysis of AGP-3 transgenic mouse Peyer's patches.
  • the Peyer's patches sections from control mouse (A, C and E) and AGP-3 transgenic mouse (B, D, and F) were stained with hematoxylin and exosin (A and B), anti-mouse B220 (C and D) or anti- mouse CD3 (E and F).
  • the histologic and immunohistologic changes were similar to the changes in the lymph node of the transgenic mouse.
  • Figure 8 shows FACS analysis of thymocytes, splenocytes and lymph node cells from AGP-3 transgenic mouse. Single-cell suspensions were prepared from spleen, lymph nodes and thymus from 10 AGP-3 transgenic mice and 5 control littermates.
  • Figure 9 shows a sequence comparison of the C-terminal region of members of the TNF ligand family determined via Pileup (Wisconsin GCG Package, Version 8.1). Amino acid numbers are indicated on the left side. The putative conserved beta strands and loops are indicated at the top. The predicted N-glycosylation sites are indicated with asterisks. The top line shows the consensus sequence (SEQ ID NO: 6). The remaining lines show the sequence for the C-terminal region of the mammalian TNF- related protein identified (SEQ ID NOS: 7 to 24, 40)
  • Figure 10 shows histology analysis of AGP-3 transgenic mice.
  • Sections of spleen (A, B, C), lymph node (D, E, F) and Payer's patches (G, H, I) from control mice (left panel) and AGP-3 transgenic mice (right panel) were stained with hematoxylin and exosin (A, D, and G), anti- mouse B220 antibody (B, E, and H), or anti-mouse CD3 antibody (C, F, and I). Stained sections were analyzed under microscope at lOx.
  • Figure 11 shows FACS analysis splenocytes, lymph node cells and thymocytes of AGP-3 transgenic mice. Single-cell suspensions were prepared form spleen, lymph nodes and thymus from 10 AGP-3 transgenic mice and 5 control littermates. Cells with stained with FTTC or PE-conjugated monoclonal antibodies against thy-1.2, B220, CDllb, Gr-1, CD4 or CD8.
  • Figure 12 shows elevation of serum immunoglobulin levels in AGP- 3 transgenic mice.
  • Serum IgM, IgG, IgA, and IgE levels were quantitated by ELISA. Values are expressed as Mean ⁇ SEM. All AGP-3 immunoglobulin levels were significantly increased (T-test; P ⁇ 0.05) compared to control groups.
  • FIG 13 shows kidney immunoglobulin deposits in AGP-3 transgenic mice. Kidney sections of 5 month control littermate (A, B, C), 5 month old AGP-3 mice (D, E, F), and 8 month old AGP-3 mice (G, H, I) were stained hematoxylin and exosin (A, D, and G), anti-mouse IgM (B, E, and H), anti-mouse IgG (C, F, and I), and Trichrome (G insert) Stained sections were analyzed under microscope at 60x. Figure 14 shows that AGP-3 stimulates B cell survival and proliferation.
  • a total of 2.5xl0 5 B cells was aliquoted per well in a 96-well round bottom plate and incubated for 9 days. At the indicated days, cells were incubated with 5 ⁇ g/ml Propidium Iodide and subject to FACS analysis for positive staining cells. Values are expressed as Mean ⁇ SEM.
  • AGP-3 stimulates B cell proliferation.
  • Purified B cells (10 5 ) from B6 mice were cultured in triplicates in 96 well plate with indicated amount of AGP-3 at the absence (upper panel) or presence of 2 ⁇ g/ml anti-IgM antibody (lower panel) for a period of 4 days. Proliferation was measured by radioactive 3 (H) thymidine uptake in last 18 hours of pulse. Data shown represent mean ⁇ standard deviation of triplicate wells.
  • AGP-3 related protein refers to natural and recombinant proteins comprising the following sequence: QDCLQLIADSXTPTIXKGXYTFVP LLSF (SEQ ID NO: 25) wherein "X” may be any naturally occurring amino acid residue. This sequence is a consensus of the B and B' ⁇ -sheets and B/B' loop of hAGP-3 and mAGP-3 (see Figure 3), which is believed to be the specific receptor binding site.
  • Preferred AGP-3-related proteins comprise both the B/B' consensus and the E/F consensus: A GHXIQRKKVHVFGDELSLVTLFR
  • the E/F region is also believed to be involved in receptor binding. More preferred proteins are those comprising the consensus of the B-I region: QDCLQLIA S XTPTIXKGXY TFVPWLLSFK RGXALEEKEN KIXVXXTGYF FIYXQVLYTD XXXAMGHXIQ RKKVHVFGDE LSLVTLFRCI QNMPXTLFN SCYSAGIAXL EEGDEXQLAI PRENAQISXX GDXTFFGALK LL (SEQ ID NO: 27)
  • AGP-3-related activity means that a natural or recombinant protein, analog, derivative or fragment is capable of modulating B cell growth, survival, or activation, particularly in MLN, spleen, and Peyer's patches.
  • Such activity can be determined, for example, by such assays as described in "Biological activity of AGP-3" in the Materials & Methods hereinafter, which may be modified as needed by many methods known to persons having ordinary skill in the art.
  • an “analog” of an AGP-3 protein is a polypeptide within the definition of "AGP-3-related protein” or "AGP-3-related protein,” respectively, that has a substitution or addition of one or more amino acids.
  • Such an AGP-3-related protein should maintain the property of eliciting B cell growth, survival, or activation.
  • Such analogs will have substitutions or additions at any place along the polypeptide.
  • Preferred analogs include those of soluble AGP-3-related proteins. Fragments or analogs may be naturally occurring, such as a polypeptide product of an allelic variant or a mRNA splice variant, or they may be constructed using techniques available to one skilled in the art for manipulating and synthesizing nucleic acids.
  • the polypeptides may or may not have an amino terminal methionine residue.
  • a “derivative" of an AGP-3 protein is a polypeptide within the definition of "AGP-3-related protein" that has undergone post- translational modifications. Such modifications include, for example, addition of N-linked or O-linked carbohydrate chains, processing of N-ter ⁇ ninal 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 of an N-terminal methionine residue due to prokaryotic host cell expression.
  • chemically modified derivatives of AGP-3-related protein that provide additional advantages such as increased stability, longer circulating time, or decreased immunogenicity are contemplated.
  • water soluble polymers such as polyethylene glycol and derivatives thereof (see for example U.S. Patent No. 4,179,337).
  • 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. Polypeptides may also be modified at pre-determined positions in the polypeptide, such as at the amino terminus, or at a selected lysine or arginine residue within the polypeptide.
  • Other chemical modifications provided include a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • protein refers to polypeptides regardless of length or origin, comprising molecules that are recombinantly produced or naturally occurring, full length or truncated, having a natural sequence or mutated sequence, with or without post-translational modification, whether produced in mammalian cells, bacterial cells, or any other expression system.
  • the invention provides for proteins referred to as AGP-3 protein, or AGP-3-related proteins that primarily act on B cells.
  • An EST bearing a portion of the AGP-3 sequence was obtained from a human fetal liver spleen cDNA library.
  • a labeled cDNA fragment was used to probe a human spleen cDNA phage library (see "Cloning of Human AGP-3" in Materials & Methods hereinafter).
  • the cDNA encoding a human AGP-3 was isolated from this phage library.
  • the human protein is a type II transmembrane protein, having a short N-terminal intracellular region that differed from other members of the TNF ligand family and a long C- terminal extracellular region that comprises most of the conserved region of the TNF ligand family.
  • An EST encoding a murine ortholog was identified by BLAST search of Genebank using the human AGP-3 sequence. The corresponding cDNA clone was obtained from a mouse lymph node library and used to probe a mouse spleen cDNA phage library (see Materials & Methods hereinafter). The cDNA encoding a murine AGP-3 ortholog was isolated from this phage library.
  • Northern blots were used to determine tissue distribution of transcription of AGP-3 (see “Cloning of Murine AGP-3" in Materials & Methods hereinafter).
  • AGP-3 mRNA was detected mainly in spleen, lung, liver, and kidney.
  • AGP-3 mRNA was detected predominantly in peripheral blood leukocytes, with weaker transcription in spleen, lung, and small intestine (see Figures 4A and 4B).
  • the murine ortholog of AGP-3 was overexpressed in transgenic mice (see “Overexpression of murine AGP-3 in transgenic mice” in Materials & Methods hereinafter).
  • mice serum globulin and total protein levels increased greatly over control littermates while the albumin level remained the same (see "Biological Activity of AGP-3" in Materials & Methods hereinafter).
  • the mice also exhibited increases in the size and number of follicles in the spleen, lymph nodes, and Peyer's patches ( Figures 5, 6, and 7).
  • the mice In their MLN, the mice exhibited 100% increases in the number of cells expressing CD45 receptor with concomitant decreases in cells expressing CD90, CD4, and CD8. These results correspond to an increase in the B cell population and a decrease in the T cell population in the MLN ( Figures 6 and 8). Similar results were obtained in the spleen, but to a lesser extent ( Figures 5 and 8).
  • nucleic acid comprises cDNA, genomic DNA, wholly or partially synthetic DNA, and RNA. These nucleic acids may be prepared or isolated as described in the working examples hereinafter or by nucleic acid hybridization thereof.
  • Nucleic acid hybridization typically involves a multi-step process.
  • a first hybridization step forms nucleic acid duplexes from single strands.
  • a second hybridization step under more stringent conditions selectively retains nucleic acid duplexes having the desired homology.
  • the conditions of the first hybridization step are generally not crucial, provided they are not of higher stringency than the second hybridization step.
  • the second hybridization is carried out under conditions of high stringency, wherein "high stringency” conditions refers to conditions of temperature and salt that are about 12-20 °C below the melting temperature (T m ) of a perfect hybrid of part or all of the complementary strands corresponding to Figure 1 (SEQ ID NO: 1) and Figure 2 (SEQ ID NO: 3).
  • high stringency conditions refer to conditions of about 65 °C and not more than about 1 M Na + . It is understood that salt concentration, temperature and /or length of incubation may be varied in either the first or second hybridization steps such that one obtains the hybridizing nucleic acid molecules according to the invention. Conditions for hybridization of nucleic acids and calculations of T m for nucleic acid hybrids are described in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, New York.
  • nucleic acids of the invention may hybridize to part or all of the polypeptide coding regions of AGP-3 related proteins (e.g., SEQ ID NOS: 2 and 4 as shown in Figures 1 and 2) and therefore may be truncations or extensions of the nucleic acid sequences shown therein. Truncated or extended nucleic acids are encompassed by the invention provided that the encoded proteins retain AGP-3 related activity.
  • the nucleic acid will encode a polypeptide of at least about 10 amino acids.
  • nucleic acid will encode a polypeptide of at least about 20 amino acids.
  • nucleic acid will encode a polypeptide of at least about 50 amino acids.
  • the hybridizing nucleic acids may also include noncoding sequences located 5' and /or 3' to the coding regions for the AGP-3 related protein.
  • Noncoding sequences include regulatory regions involved in expression of AGP-3 related protein, such as promoters, enhancer regions, translational initiation sites, transcription termination sites and the like.
  • the nucleic acids of the invention encode mouse or human AGP-3. Most preferred are the nucleic acids encoding proteins of SEQ ID NOS: 25, 26, or 27. Nucleic acids may encode a membrane-bound form of AGP-3-related protein or soluble forms.
  • the predicted transmembrane region includes amino acid residues 47-72 inclusive as shown in Figure 1 (SEQ. ID. NO: 2); for murine AGP-3 related protein, residues 48-73 inclusive as shown in Figure 2 (SEQ ID NO: 4). Substitutions that replace hydrophobic amino acid residues in this region with neutral or hydrophilic amino acid residues would be expected to disrupt membrane association and result in soluble AGP-3-related protein.
  • Nucleic acid sequences of the invention may also be used for the detection of sequences encoding AGP-3-related protein in biological samples.
  • the sequences may be used to screen cDNA and genomic libraries for related AGP-3-related protein sequences, especially those from other species.
  • the nucleic acids are also useful for modulating levels of AGP-3-related protein by anti-sense technology or in vivo gene expression. Development of transgenic animals expressing AGP-3-related protein are useful for production of the polypeptides and for the study of in vivo biological activity.
  • nucleic acids of the invention will be linked with DNA sequences so as to express biologically active AGP-3-related protein.
  • Sequences required for expression are known to those skilled in the art and include promoters and enhancer sequences for initiation of RNA synthesis, transcription termination sites, ribosome binding sites for the initiation of protein synthesis, and leader sequences for secretion.
  • Sequences directing expression and secretion of AGP-3-related protein may be homologous, i.e., the sequences are identical or similar to those sequences in the genome involved in AGP-3-related protein expression and secretion, or they may be heterologous.
  • plasmid vectors are available for expressing AGP-3-related protein in host cells (see, for example, Methods in Enzymology v. 185, Goeddel, D.V. ed., Academic Press (1990)).
  • plasmid pDSR ⁇ described in PCT Application No. 90/14363.
  • preferred embodiments include plasmids harboring the lux promoter (see co-owned and co-pending U.S. Serial No. 08/577,778, filed December 22, 1995).
  • vectors are available for the tissue-specific expression of AGP-3- related protein in transgenic animals.
  • Gene transfer vectors derived from retrovirus (RV), adenovirus (AdV), and adeno-associated virus (AAV) may also be used for the expression of AGP-3 related protein in human cells for in vivo therapy (see PCT Application No.86/00922).
  • Prokaryotic and eukaryotic host cells expressing AGP-3-related protein are also provided by the invention.
  • Host cells include bacterial, yeast, plant, insect or mammalian cells.
  • AGP-3-related protein may also be produced in transgenic animals, such as mice or goats. Plasmids and vectors contaii ⁇ rtg the nucleic acids of the invention are introduced into appropriate host cells using transfection or transformation techniques known to one skilled in the art.
  • Host cells may contain DNA sequences encoding AGP-3-related protein as shown in Figures 1, 2, or 3, or a portion of either thereof, such as the extracellular domain or the cytoplasmic domain.
  • Nucleic acids encoding AGP-3-related proteins may be modified by substitution of codons that allow for optimal expression in a given host. At least some of the codons may be so-called preference codons that do not alter the amino acid sequence and are frequently found in genes that are highly expressed. However, it is understood that codon alterations to optimize expression are not restricted to the introduction of preference codons.
  • Examples of preferred mammalian host cells for AGP- 3-related protein expression include, but are not limited to COS, CHOd-, 293 and 3T3 cells.
  • a preferred bacterial host cell is Escherichia coli.
  • the invention also provides AGP-3-related proteins as the products of prokaryotic or eukaryotic expression of exogenous DNA sequences.
  • Exogenous DNA sequences include cDNA, genomic DNA and synthetic DNA sequences.
  • AGP-3-related proteins may be the products of bacterial, yeast, plant, insect or mammalian cells expression, or from cell-free translation systems.
  • AGP-3-related proteins produced in bacterial cells will have N-te ⁇ rtinal methionine residues.
  • the invention also provides for a process of producing AGP-3-related proteins comprising growing prokaryotic or eukaryotic host cells transformed or transfected with nucleic acids encoding them and isolating polypeptide expression products of the nucleic acids.
  • polypeptides that are mammalian proteins or are fragments, analogs or derivatives thereof are encompassed by the invention.
  • the AGP-3-related protein is human AGP-3 protein.
  • a fragment of AGP-3-related protein refers to a polypeptide having a deletion of one or more amino acids such that the resulting polypeptide retains AGP-3 related activity; for example, the polypeptide has at least the property of eliciting or antagonizing B cell growth, survival, or activation, especially in mesenteric lymph nodes. Said fragments will have deletions originating from the amino terminal end, the carboxy terminal end, and internal regions of the polypeptide.
  • Fragments of AGP-3-related proteins are at least about ten amino acids, at least about 20 amino acids, or at least about 50 amino acids in length.
  • AGP-3-related proteins will have a deletion of one or more amino acids from the transmembrane region (amino acid residues 48-73 as shown in Figure 1), or, alternatively, one or more amino acids from the ammo-terminus up to and /or including the transmembrane region (amino acid residues 1-73 as shown in Figure 1).
  • Such polypeptides may act as agonists or antagonists of the ligand :receptor interaction and activate or inhibit ligand-mediated activity of AGP-3 related protein.
  • Such antagonists and /or agonists can be examined for AGP-3 related activity (see "Biological activity of AGP-3" in Materials & Methods hereinafter).
  • polypeptides of the invention are isolated and purified from tissues and cell lines that express AGP-3 related protein, either extracted from lysates or from conditioned growth medium, and from transformed host cells expressing AGP-3 related protein.
  • Human AGP-3 related protein, or nucleic acids encoding same, may be isolated from human lymph node or fetal liver tissue. Isolated AGP-3 related protein is free from association with human proteins and other cell constituents.
  • a method for purification of such proteins from natural sources e.g. tissues and cell lines that normally express an AGP-3 related protein
  • the purification process may employ one or more standard protein purification steps in an appropriate order to obtain purified protein.
  • the chromatography steps can include ion exchange, gel filtration, hydrophobic interaction, reverse phase, chromatofocusing, affinity chromatography employing an anti-AGP-3-related protein antibody or biotin-streptavidin affinity complex and the like.
  • Fusion proteins and derivatives The invention further comprises AGP-3-related protein chimeras, as well as such proteins derivatized by linkage to such molecules as PEG or dextran.
  • Such proteins comprise part or all of an AGP-3-related protein amino acid sequence fused to a heterologous amino acid sequence.
  • the heterologous sequence may be any sequence that allows the resulting fusion protein to retain AGP-3-related activity (i.e., AGP-3 agonists) or will maintain AGP-3 binding activity but not have AGP-3 related activity as defined herein (i.e., AGP-3 antagonists).
  • AGP-3-related activity i.e., AGP-3 agonists
  • AGP-3 antagonists i.e., AGP-3 antagonists.
  • fragments, derivatives or analogs of AGP-3 can be examined for their ability to agonize or antagonize AGP-3-mediated B cell growth, survival, or activation associated with the disease or condition (see "Biological activity of AGP-3" in Materials & Methods hereinafter).
  • a heterologous sequence is fused to a sequence comprising an AGP-3 related protein's B/B' region (SEQ ID NO: 25) and/or the E/F region (SEQ ID NO: 26) or to the more complete B-I region (SEQ ID NO: 27).
  • heterologous sequences include cytoplasmic domains that allow for alternative intracellular signaling events, sequences that promote oligomerization (e.g., the Fc region of IgG), enzyme sequences that provide a label for the polypeptide, and sequences that provide affinity probes (e.g., an antigen-antibody recognition site).
  • Preferred molecules in accordance with this invention are Fc-linked
  • the AGP-3 related protein may be attached to a vehicle through the protein's N-terminus or C-terminus.
  • vehicle-protein molecules of this invention may be described by the following formula I:
  • F 1 is a vehicle (preferably an Fc domain);
  • X 1 and X 2 are each independently selected from -(L ⁇ -P 1 , -(L ⁇ -P 1 -
  • P 1 , P 2 , P 3 , and P 4 are each independently sequences of AGP-3 related protein (e.g., a fragment of hAGP-3); L 1 , L 2 , L 3 , and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.
  • AGP-3 related protein e.g., a fragment of hAGP-3
  • L 1 , L 2 , L 3 , and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.
  • compound I comprises preferred compounds of the formulae ⁇ X'-F 1 and multimers thereof wherein F 1 is an Fc domain and is attached at the C- terminus of X 1 ; nr
  • antibodies specifically binding the polypeptides of the invention are also encompassed by the invention.
  • the antibodies may be generated by immunization with full-length AGP-3 related protein, or fragments thereof.
  • Preferred antibodies bind to SEQ ID NOS: 25, 26, or 27.
  • Such antibodies may be generated by immunization with polypeptides comprising those sequences.
  • the term "antibodies” also refers to molecules having Fv, Fc and other structural domains usually associated with antibodies but that may be generated by other techniques (e.g., phage display antibody generation).
  • the antibodies of the invention may be polyclonal or monoclonal, or may be recombinant antibodies, such as chimeric antibodies wherein the murine constant regions on light and heavy chains are replaced by human sequences, or CDR-grafted antibodies wherein only the complementarity determining regions are of murine origin.
  • Antibodies of the invention may also be fully human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Application No. W093/ 12227). Regardless of the means by which they are generated, antibodies in accordance with this invention may be produced by recombinant means (e.g., transfection of CHO cells with vectors comprising antibody sequence).
  • the antibodies are useful for detecting AGP-3 related protein in biological samples, thereby allowing the identification of cells or tissues that produce such proteins.
  • antibodies that bind to AGP-3 related proteins and block interaction with other binding compounds i.e., "antagonist antibodies”
  • antibodies can be tested for binding to AGP-3 related protein and examined for their effect on AGP-3- mediated B cell growth, survival, or activation associated with the disease or condition (see "Biological activity of AGP-3" in Materials & Methods hereinafter).
  • compositions comprising a therapeutically effective amount of the AGP-3 related protein of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • pharmaceutical compositions comprising a therapeutically effective amount of an AGP-3 related protein agonist or antagonist.
  • therapeutically effective amount means an amount that provides a therapeutic effect for a specified condition and route of adrninistration.
  • the composition may be in a liquid or lyophilized form and comprises one or more of the following: • a diluent (e.g., Tris, acetate or phosphate buffers) having various pH values and ionic strengths;
  • solubilizer e.g., Tween or Polysorbate
  • carriers e.g., human serum albumin or gelatin
  • preservatives e.g., thimerosal or benzyl alcohol
  • antioxidants e.g., ascorbic acid or sodium metabisulfite
  • compositions comprising AGP-3 antibody or soluble AGP-3-related protein are provided. Also encompassed are compositions comprising soluble AGP-3-related protein modified with water-soluble polymers to increase solubility, stability, plasma half-life and bioavailability. Compositions may also comprise incorporation of soluble AGP-3 related protein into liposomes, microemulsions, micelles or vesicles for controlled delivery over an extended period of time. Soluble AGP-3 related protein may be formulated into microparticles suitable for pulmonary administration. Compositions of the invention may be administered by injection (either subcutaneous, intravenous or intramuscular) or by oral, nasal, pulmonary or rectal administration. The route of administration eventually chosen will depend upon a number of factors and may be ascertained by one of ordinary skill in the art.
  • the invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of the nucleic acids of the invention together with a pharmaceutically acceptable adjuvant.
  • Nucleic acid compositions will be suitable for the delivery of part or all of the coding region of AGP-3 related protein and/or flanking regions to cells and tissues as part of an anti-sense therapy regimen.
  • AGP-3 related proteins and agonists or antagonists thereof may be used to treat conditions characterized by B cell growth, survival, and activation, such as autoimmune and inflammatory disorders.
  • the invention also encompasses modulators (agonists and antagonists) of AGP-3-related protein and methods for obtaining them.
  • modulator may either increase or decrease at least one activity associated with AGP- 3, such as B cell growth, survival, or activation in MLN, spleen, and Peyer's patches.
  • an agonist or antagonist may be a co-factor, such as a protein, peptide, carbohydrate, lipid or small molecular weight molecule, that interacts with AGP-3 and regulates activity.
  • Potential polypeptide antagonists include antibodies that react with soluble or membrane-associated forms of AGP-3, a fragment of AGP-3 (e.g., SEQ ID NO: 25) and an Fc-linked AGP-3 fragment.
  • Molecules that regulate AGP- 3-related protein expression typically include nucleic acids that are complementary to nucleic acids encoding AGP-3-related protein and that act as anti-sense regulators of expression.
  • AGP-3-related proteins and modulators thereof may be particularly useful in treatment of inflammatory conditions of the joints.
  • Inflammatory conditions of a joint are chronic joint diseases that afflict and disable, to varying degrees, millions of people worldwide.
  • Rheumatoid arthritis is a disease of articular joints in which the cartilage and bone are slowly eroded away by a proliferative, invasive connective tissue called pannus, which is derived from the synovial membrane.
  • the disease may involve peri-articular structures such as bursae, tendon sheaths and tendons as well as extra-articular tissues such as the subcutis, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) and eyes (Silberberg (1985), Anderson's Pathology, Kissane (ed.), 11:1828).
  • Osteoarthritis is a common joint disease characterized by degenerative changes in articular cartilage and reactive proliferation of bone and cartilage around the joint. Osteoarthritis is a cell-mediated active process that may result from the inappropriate response of chondrocytes to catabolic and anabolic stimuli.
  • AGP-3 related proteins and agonists or antagonists thereof may also be useful in treatment of a number of additional diseases and disorders, including: acute pancreatitis; ALS;
  • Alzheimer's disease Alzheimer's disease; asthma; atherosclerosis; cachexia / anorexia; chronic fatigue syndrome; diabetes (e.g., insulin diabetes); fever; glomerulonephritis; graft versus host disease; hemorrhagic shock; hyperalgesia; inflammatory bowel disease; inflammatory conditions of a joint, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; inflammatory conditions resulting from strain, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease processes; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration); learning impairment; lung diseases (e.g., ARDS); multiple myeloma; multiple sclerosis; myelogenous leukemia (e.g., AML and CML) and other leukemias; myopathies (e.g., muscle protein metabolism, esp.
  • Parkinson's disease pre-term labor; psoriasis; reperfusion injury; septic shock; side effects from radiation therapy; sleep disturbance; temporal mandibular joint disease; and tumor metastasis.
  • Agonists and antagonists of AGP-3-related protein may be administered alone or in combination with a therapeutically effective amount of other drugs, including analgesic agents, disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and any immune and /or inflammatory modulators.
  • DMARDs disease-modifying anti-rheumatic drugs
  • NSAIDs non-steroidal anti-inflammatory drugs
  • agonists and antagonists of AGP-3 related protein may be administered with:
  • TNF antagonists such as etanercept (EnbrelTM), sTNF- Rl, D2E7, and RemicadeTM.
  • NGF Nerve growth factor
  • IL-1 inhibitors including IL-lra molecules such as anakinra (KineretTM) and more recently discovered IL-lra-like molecules such as IL-lHyl and IL-lHy2; IL-1 "trap" molecules as described in U.S. Pat. No. 5,844,099, issued December 1, 1998; IL-1 antibodies; solubilized IL-1 receptor, and the like.
  • IL-lra molecules such as anakinra (KineretTM) and more recently discovered IL-lra-like molecules such as IL-lHyl and IL-lHy2
  • IL-1 "trap" molecules as described in U.S. Pat. No. 5,844,099, issued December 1, 1998
  • IL-1 antibodies solubilized IL-1 receptor, and the like.
  • IL-6 inhibitors e.g., antibodies to IL-6.
  • IL-8 inhibitors e.g., antibodies to IL-8.
  • IL-18 inhibitors e.g., IL-18 binding protein, solubilized IL-18 receptor, or IL-18 antibodies.
  • Interleukin-1 converting enzyme (ICE) modulators Interleukin-1 converting enzyme (ICE) modulators. insulin-like growth factors (IGF-1, IGF-2) and modulators thereof.
  • IGF-1 insulin-like growth factors
  • IGF-2 insulin-like growth factors
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ family members Transforming growth factor- ⁇ (TGF- ⁇ ), TGF- ⁇ family members, and TGF- ⁇ modulators.
  • Fibroblast growth factors FGF-1 to FGF-10 Fibroblast growth factors FGF-1 to FGF-10, and FGF modulators.
  • Osteoprotegerin OPG
  • OPG osteoprotective agents
  • bone anabolic agents osteoprotective agents
  • KGF Keratinocyte growth factor
  • KGF-2 KGF-2
  • KGF modulators KGF-2
  • COX-2 inhibitors such as CelebrexTM and VioxxTM.
  • Prostaglandin analogs e.g., E series prostaglandins.
  • MMP Matrix metalloproteinase
  • Nitric oxide synthase (NOS) modulators including modulators of inducible NOS.
  • LPS lipopolysaccharide
  • Anti-cancer agents including inhibitors of oncogenes (e.g., fos, jun) and interferons.
  • AGP-3-related proteins may be used in a variety of assays for detecting agonists, antagonists and characterizing interactions with AGP-3 related proteins.
  • the assay comprises incubating AGP-3-related protein under conditions that permit measurement of AGP-3-related activity as defined above.
  • Qualitative or quantitative assays may be developed.
  • Assays may also be used to identify new AGP-3 agonists or antagonists and AGP-3 related potein family members.
  • Binding assays for agonists, or antagonists to natural or recombinant AGP-3 related protein may be carried out in several formats, including cell-based binding assays, membrane binding assays, solution- phase assays and imrnunoassays.
  • trace levels of a labeled binding molecule are incubated with AGP-3-related protein samples for a specified period of time followed by measurement of bound molecule by filtration, electrochemiluminescent (ECL, ORIGEN system by IGEN), cell- based or immunoassays.
  • ECL electrochemiluminescent
  • Homogeneous assay technologies for radioactivity (SPA; Amersham) and time-resolved fluoresence (HTRF, Packard) can also be implemented.
  • Binding is detected by labeling a binding molecule (e.g., an anti-AGP-3 antibody) with radioactive isotopes (1251, 35S, 3H), fluorescent dyes (fluorescein), lanthanide (Eu 3* ) chelates or cryptates, orbipyridyl-ruthenium (Ru 2+ ) complexes.
  • a binding molecule may be modified with an unlabeled epitope tag (e.g., biotin, peptides, His 6 , myc) and bound to proteins such as streptavidin, anti-peptide or anti-protein antibodies that have a detectable label as described above.
  • Binding molecules in such assays may be nucleic acids, proteins, peptides, carbohydrates, lipids or small molecular weight organic compounds.
  • the binding molecule may be substantially purified or present in a crude mixture.
  • the binding molecules may be further characterized by their ability to increase or decrease AGP-3 related activity in order to determine whether they act as an agonist or an antagonist.
  • AGP-3-related protein may be assayed directly using polyclonal or monoclonal antibodies to AGP-3 related proteins in an immunoassay. Additional forms of AGP-3-related proteins containing epitope tags as described above may be used in solution and immunoassays.
  • AGP-3 related proteins are also useful for identification of intracellular proteins that interact with their respective cytoplasmic domains by a yeast two-hybrid screening process.
  • hybrid constructs comprising DNA encoding the N-te ⁇ riinal 50 amino acids of an AGP-3 related protein fused to a yeast GAL4-DNA binding domain may be used as a two-hybrid bait plasmid. Positive clones emerging from the screening may be characterized further to identify interacting proteins.
  • This information may help elucidate an intracellular signaling mechanism associated with AGP-3-related activity and provide intracellular targets for new drugs that modulate inflammatory and immune-related diseases and conditions.
  • a variety of assays may be used to measure the interaction of AGP-
  • AGP-3 related protein can be immobilized by attachment to the bottom of the wells of a microtiter plate.
  • a radiolabeled binding molecule and a test molecule can then be added either one at a time (in either order) or simultaneously to the wells. After incubation, the wells can be washed and counted using a scintillation counter for radioactivity to determine the extent of binding to AGP-3 related protein.
  • molecules will be tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assays can be used for accuracy in evaluation of the results.
  • An alternative to this method involves reversing the "positions" of the proteins; i.e., immobilizing a binding molecule to the mictrotiter plate wells, incubating with the test compound and radiolabeled AGP-3 related protein, and determining the extent of binding. See, for example, chapter 18 of Current Protocols in Molecular Biology (1995) (Ausubel et al., eds.), John Wiley & Sons, New York, NY.
  • AGP-3 related proteins or a binding molecule may be conjugated to biotin and the presence of biotinylated protein can then be detected using streptavidin linked to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), that can be detected colorimetrically, or by fluorescent tagging of streptavidin.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • An antibody directed to AGP-3 related protein or a binding molecule that is conjugated to biotin may also be used and can be detected after incubation with enzyme-linked streptavidin linked to AP or HRP.
  • AGP-3-related proteins or binding molecules may also be immobilized by attachment to agarose beads, acrylic beads or other types of such inert substrates.
  • the substrate-protein complex can be placed in a solution containing the complementary protein and the test compound; after incubation, the beads can be precipitated by centrifugation, and the amount of binding between the AGP-3-related protein and a binding molecule can be assessed using the methods described above.
  • the substrate-protein complex can be immobilized in a column and the test molecule and complementary molecule passed over the column. Formation of a complex between AGP-3 related protein and the binding molecule can then be assessed using any of the techniques set forth above (i.e., radiolabeling, antibody binding, and the like).
  • Biacore assay is a surface plasmon resonance detector system, such as the Biacore assay system (Pharmacia, Piscataway, NJ).
  • the Biacore system may be carried out using the manufacturer's protocol.
  • This assay essentially involves covalent binding of either an AGP-3 related protein or a binding molecule to a dextran-coated sensor chip that is located in a detector.
  • the test compound and the other complementary protein can then be injected into the chamber containing the sensor chip either simultaneously or sequentially and the amount of complementary protein that binds can be assessed based on the change in molecular mass that is physically associated with the dextran-coated side of the of the sensor chip; the change in molecular mass can be measured by the detector system.
  • In vitro assays such as those described above may be used advantageously to screen rapidly large numbers of compounds for effects on complex formation with AGP-3-related proteins.
  • the assays may be automated to screen compounds generated in phage display, synthetic peptide and chemical synthesis libraries.
  • Compounds that increase or decrease complex formation of AGP-3- related proteins and binding molecules may also be screened in cell culture using cells and cell lines bearing such ligands.
  • Cells and cell lines may be obtained from any mammal, but preferably will be from human or other primate, canine, or rodent sources. Such cells may be enriched from other cell types by affinity chromatography using publicly available procedures. Attachment of AGP-3-related protein to such cells is evaluated in the presence or absence of test compounds and the extent of binding may be determined by, for example, flow cytometry using a biotinylated antibody. Cell culture assays may be used advantageously to further evaluate compounds that score positive in protein binding assays described above. Description of Preferred Embodiments
  • a TNF family profile search of the Genbank dbEST data base was performed. Smith et al.(1994), Cell, 76: 959-62; Luethy etal.(1994), Protein Science, 3: 139-46.
  • One human EST sequence (GenBank accession number T87299) was identified as a possible new member of the TNF ligand. The EST was obtained from human fetal liver spleen cDNA library (The
  • the cDNA clone (115371 3') corresponding to the EST sequence was obtained from Genome Systems, Inc. (St. Louis, MO).
  • the cDNA fragment was released from the pT7T3D vector with EcoRI and Notl digestion. The fragment was approximately 0.7 kb in length and was used for the subsequent full-length cloning.
  • the 32 P-dCTP-labeled T87299 cDNA fragment was used as a probe to screen a human spleen cDNA phage library (Stratagene, La Jolla, CA). Recombinant phages were plated onto E. coli strain XLl-blue at approximately 5 x 10 4 transformants per 150 mm LB plate. Nitrocellulose filters were lifted from these plates in duplicates. Filters were prehybridized in 5x SSC, 50% deionized formamide, 5x Denhardt's solution, 0.5% SDS, and 100 ⁇ g/ml denatured salmon sperm DNA for 2 hours at 42 °C.
  • the filters were then hybridized in the same solution with the addition of 5 ng/ml of labeled probe at 42°C overnight.
  • the filters were first washed in 2x SSC and 0.1% SDS for 10 minutes at RT twice, and then washed in O.lx SSC and 0.1% SDS at 65 °C for 30 minutes twice.
  • the filters were then exposed to autoradiography with intensifying screens at - 80 °C overnight. Positive hybridizing plaques were determined by aligning the duplicate filters, and then picked up for subsequent secondary or tertiary screening till single isolated positive plaque was obtained. From total of one million recombinant phage clones, 8 positive plaques were obtained.
  • the pBluescript phagemid was excised from phage using the ExAssistTM/SOLRTM System according to the manufacturer's description (Stratagene, La Jolla, CA). The excised phagemids were plated onto freshly grown SOLR cells on LB/ampicillin plates and incubated overnight. Single bacteria colony was amplified in LB media containing 100 ⁇ g/ml ampicillin. The plasmid DNA was prepared and both strands of cDNA insert were sequenced.
  • human AGP-3 cDNA (clone 13-2) is 1.1 kb in length. It encodes a LORF of 285 amino acids. FASTA search of the SwissProt database with the predicted AGP-3 protein sequence indicated that it is mostly related to human TNF ⁇ with 25% identity in C-terminal 116 amino acid overlap.
  • human AGP-3 protein is a type II transmembrane protein, containing a short N-terminal intracellular domain (amino acids 1-46), a hydrophobic transmembrane region (amino acids 47-68) following by a long C-terminal extracellular domain (amino acid 69-285). The C-terminal extracellular domain of AGP-3 contained most of the conserved region of the TNF ligand family. Smith et al.(1994), Cell, 76: 959-62.
  • EST sequence (Genebank accession number AA254047) encoding a potential murine AGP-3 ortholog was identified by BLAST search of Genebank dbEST database with human AGP-3 sequence.
  • the corresponding cDNA clone (722549 5') from mouse lymph node library was obtained from Genome Systems, Inc. (St. Louis, MO).
  • the clone contained a 0.9 kb cDNA insert which could be released by EcoRI and Notl digestion.
  • the 0.9 kb cDNA fragment encodes an open reading frame of 96 amino acids which shares 87% identity with the corresponding C- terminal human AGP-3 polypeptide sequence.
  • a 0.41 kb EcoRI-Xmnl fragment which contained 290 bp coding region and 120 bp 3' non-coding region, was used as probe to screening a mouse spleen cDNA phage library (Stratagene, La Jolla, CA) for full length murine AGP-3 cDNA as described above. From one million recombinant phage clones, 6 positive plaques were obtained. The phagemid was excised from phage as described above. The plasmid DNA was prepared and both strands of cDNA insert were sequenced. The murine AGP-3 cDNA (clone S6) encodes a polypeptide of 309 amino acids.
  • murine AGP-3 is also a type II transmembrane region, containing a short N-terminal intracellular domain (amino acid 1 ⁇ 46), a hydrophobic transmembrane region (amino acid 47-68) following by a long C-terminal extracellular domain (amino acid 69-285).
  • the human and murine AGP-3 share 68% amino acid sequence identity overall. However, the C-terminal 142 amino acid sequences share 87% identity between the two species. Preceding the highly conserved C-terminus region, there is an insertion of 30 extra amino acids in the murine AGP-3. Four out of 7 positive phage plaques were independent clones, yet they all shared the same coding sequences.
  • Murine AGP-3 cDNA clone S6 in pBluescript SK(-) in pBluescript was used as template to PCR the entire coding region.
  • T3 primer 5' AAT TAA CCC TCA CTA AAG GG 3" SEQ ID NO: 28 was used as 5' PCR primer.
  • SEQ ID NO: 29 PCR reactions were carried in a volume of 50 ⁇ l with 1 unit of vent DNA polymerase (New England Biolabs) in 20 mM Tris-HCl pH 8.8, 10 mM (NH -SO ⁇ 0.1% Triton-XlOO, 10 ⁇ M of each dNTP, 1 ⁇ M of each primer and 10 ng of murine AGP-3 cDNA template. Reactions were performed in 94 °C for 45 s, 55 °C for 55 S, and 72 °C for 2 minutes, for a total of 35 cycles. The PCR fragment created a Xhol site at 3' end after the AGP-3 coding region.
  • the 1 kb PCR fragment was purified by elecrrophoresis, and digested with Xbal (present in the pBluescript MCS, 80 bp upstream of AGP-3 starting Methione) and Xhol restriction enzymes.
  • the Xbal-Xhol PCR fragment was cloned into expression vector under the control of the human ⁇ -actin promoter. Graham et al.(1997), Nature Genetics 17: 272-4; Ray et_al.(1991), Genes Dev. 5: 2265-73.
  • the PCR fragment was sequenced to ensure no mutation.
  • the murine AGP-3 expression plasmid was purified through two rounds of CsCl density gradient centrifugation.
  • the purified plasmid was digested with Clal, and a 6 kb fragment containing murine AGP-3 transgene was purified by gel electrophoresis. The purified fragment was resuspended in 5 mM Tris, pH 7.4, 0.2 mM EDTA at 2 ⁇ g/ml concentration.
  • Single-cell embryos from BDF1 x BDFl-bred mice were injected as described (W097 / 23614). Embryos were cultured overnight in a CO- incubator and 15-202-cell embryos were transferred to the oviducts of pseudopregnant CD1 female mice.
  • offspring were obtained from implantation of microinjected embryos.
  • the offspring were screened by PCR amplification of the integrated transgene in genomic DNA samples. Ear pieces were digested in 20 ⁇ l ear buffer (20mM Tris, pH8.0, lOmM EDTA, 0.5% SDS, 500 ⁇ g/ml proteinase K) at 55°C overnight. The sample was diluted with 200 ⁇ l of TE, and 2 ⁇ l of the ear sample was used for the PCR reaction.
  • the PCR reactions were carried in a volume of 50 ⁇ l with 0.5 unit of Tag DNA polymerase (Boehringer Mannheim, Indianapolis, IN) in 10 mM Tris-HCl pH 8.3, 50 mM KC1, 2.5 mM MgCl 2 , 10 ⁇ M of each dNTP, 1 ⁇ M of each primer and 2 ⁇ l of ear sample.
  • Tag DNA polymerase Boehringer Mannheim, Indianapolis, IN
  • the mixtures were first heated at 94 °C for 2 min, and the PCR reactions were performed in 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, for a total of 35 cycles.
  • 10 were identified as PCR positive transgenic founders.
  • RNA were digested by addition of 2 U RNase H and incubation at 37 °C for 20 min.
  • Subsequent PCR reactions were carried out by using murine AGP-3 specific primers.
  • the 5' PCR primer was 5' AGC CGC GGC CAC AGG AAC AG 3'
  • the PCR reaction was performed in a volume of 50 ⁇ l with 0.5 unit Tag DNA polymerase in 10 mM Tris-HCl pH 8.3, 50 mM KCL, 2.5mM MgCl 2 , 10 ⁇ M of each dNTP, 1 ⁇ M of each primer and 1 ⁇ l of cDNA product.
  • the reaction was performed at 94 °C for 30 s, 55°C for 30 S, and 72 °C for 1 min, for a total of 35 cycles.
  • the PCR product was analyzed by electrophoresis. Transgene expression was detected in the spleen of all ten AGP-3 transgenic mice founders.
  • Radiography was performed after terminal exsanguination. There was no difference in the radiodensity or radiologic morphology of the skeleton. Upon gross dissection, major visceral organs were subject to weight analysis. The spleen weight relative to the body weight increased by approximately 45% in the AGP-3 transgenic group as compared to the control mice. The sizes of lymph nodes and Peyer's patches were also increased substantially in all the AGP-3 transgenic mice. Following gross dissection, tissues were removed and fixed in 10% buffered Zn-Formalin for histological examination.
  • the tissues collected were liver, spleen, pancreas, stomach, the entire gastrointestinal tract, kidney, reproductive organs, skin and mammary glands, bone, brain, heart, lung, thymus, trachea, esophagus, thyroid, adrenals, urinary bladder, lymph nodes and skeletal muscle. After fixation, the tissues were processed into paraffin blocks, and 3 ⁇ m sections were obtained. All sections were stained with hematoxylin and exosin, and subject to histologic analysis. The size and the number of the follicles in the spleen, lymph nodes and Peyer's patches were increased significantly in the AGP- 3 transgenic mice ( Figure 5, 6 and 7).
  • the spleen, lymph node and Peyer's patches of both the transgenic and the control mice were subject to immunohistology analysis with B cell and T cell specific antibodies.
  • the formalin fixed paraffin embedded sections were deparaffinized and hydrated to deionized water. The sections were quenched with 3% hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh, PA), and incubated in rat monoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, IN), respectively.
  • the binding was detected by biotinylated rabbit anti-rat immunoglobulins and peroxidase conjugated streptavidin (BioGenex, San Ramon, CA) with DAB as chromagen (BioTek, Santa
  • the B cell numbers as indicated by positive B220 staining, increased significantly in the spleen, lymph nodes and Peyer's batches ( Figure 5, 6, and 7).
  • the T cell numbers as indicated by the anti-CD3 staining, were slightly decreased. There were no differences in the morphology of the thymus between the transgenic and the control group. By immunohistology, the T cell population was similar in numbers. At 8 weeks of age, there are no distinctive morphologic changes in the liver, kidneys, or urinary, central nervous, hematopoietic, skeletal, respiratory, gastrointestinal, endocrine, or reproductive systems.
  • MLN and sections of spleen and thymus from 10 AGP-3 transgenic mice animals 3, 6, 9, 10, 13, 38, 40, 58, 59 and 62
  • 5 control littermates animals 7, 8, 11, 12, and 14
  • Single cell suspensions were prepared by gently grinding the tissues with the flat end of a syringe against the bottom of a 100 ⁇ m nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Cells were washed twice in a 15 ml volume then counted.
  • the cells were washed then analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA). Thymus samples were stained with F TC conjugated anti-Thy-1.2, F TC conjugated anti-CD4, and PE conjugated anti-CD8 (PharMingen, San Diego, CA).
  • Serum Immunoglobulin and Autoantibody Analysis Transgenic mice and control littermates were bled successively at 6, 7, 8, 9, 11, and 12 weeks of age. Serum immunoglobulin levels were quantitated using by ELISA with Mouse Hybridoma Subtype Kit as suggested by manufacture (Boehringer Mannheim, Indianapolis, IN). Presence of autoantibodies directed against nuclear antigens and dsDNA were examined in the serum by enzyme linked immunosorbant assay (ELISA). The levels of anti-nuclear antibodies were detected using ANA screen kit (Sigma) and anti-mouse IgG peroxidase secondary antibody. Mouse serum samples were diluted 1:200 in ANA screen ELISA.
  • B lymphocytes were isolated from spleens of 2-4 months old mice by negative selection. Briefly, B lymphocytes were purified by density gradient centrifugation and then passed over a B cells column (Accurate/ Cedarlane, Westbury, NY). Cells isolated by this method were analyzed by flow cytometry and >90% were found positive for B220 staining. Isolated B cells were cultured in MEM+10% FCS at 37°C, 5%C0 2 . Cells were collected from triplicate wells daily on day 1 through day 9 and incubated with 5 ⁇ g/ml Propidium Iodide. Cells were analyzed by Flow cytometry and the percentage of dead cells was calculated.
  • B cell proliferation assay purified (10 5 ) B cells from B6 mice as described above were cultured in MEM+10% heat inactivated FCS in triplicate in 96 well flat bottomed plate with/without 2 ⁇ g/ml of Goat F(ab') 2 anti-mouse IgM (Jackson ImmunoResearch Laboratory, West Grove, Pennsylvania) and /or indicated amount of recombinant AGP-3 for a period of 4 days at 37 °C, 5%C0 2 . Proliferation was measured by an uptake of radioactive 3 (H) thymidine in last 18 hours of pulse. Data is shown in figure 14 as mean+standard deviation of triplicate wells. -3; 260-3. B Cell Hyperplasia and Hyperga maglobulinemia in AGP-3
  • transgenic mice were generated that expressed full-length murine AGP-3 protein driven by the ubiquitous 3-actin promoter. Founder mice harboring the AGP-3 transgene were identified by PCR analysis of genomic DNA samples. Transgene expression was confirmed by RT-PCR from spleen total RNA.
  • the transgenic mice were of normal size and weight. However, the spleen weight relative to the body weight increased by approximately 45% in the AGP-3 transgenic group as compared to the control mice. The sizes of lymph nodes and Payer's Patches were also increased substantially in all the AGP-3 transgenic mice. Histology analysis demonstrated that the size and the number of the follicles in the spleen, lymph nodes and Payer's patches were increased significantly in the AGP-3 transgenic group
  • the B cell hyperplasia phenotype in the AGP-3 transgenic mice was also confirmed by flow cytometry analysis.
  • the percentage of B220 positive B cells increased by 100% (Figure 11).
  • the percentage of the Thy-1.2 positive T cells decreased by approximately 36%, with similar reductions in both CD4(+) and CD8(+) T cells.
  • Similar increase in B cell and reduction in T cell populations were also observed in the spleens of the AGP-3 transgenic mice, though to a lesser extent ( Figure 11).
  • the total T cell numbers in the lymph node and spleen of AGP-3 transgenic mice were similar to the control littermates.
  • the AGP-3 transgenic mice In addition to the B cell hyperplasia phenotype, the AGP-3 transgenic mice also had severe hypergammaglobulinemia .
  • the serum globulin level in AGP-3 transgenic mice increased more that 100% as compared to the control group. Total protein level also increased correspondingly in the transgenic, while albumin level remained the same.
  • the increased B cell numbers and high serum globulin level suggested elevated serum immunoglobulin titer.
  • serum levels of IgM, IgG, IgA and IgE of AGP-3 transgenic mice from 6 to 12 weeks of age. Comparing to the same age control littermates, serum IgM, IgG, IgA and IgE were significantly increased in all age groups of AGP-3 transgenic mice.
  • the B cell hyperplasia phenotype in the AGP-3 transgenic mice might arise from increased B cell survival and /or increased B cell proliferation.
  • B cells were isolated from both transgenic or control mice and incubated in minimal essential medium supplemented with 10% heat inactivated fetal bovine serum. Viability of the B cells was measured by FACS analysis for Propidium Iodide uptake (Figure 14A). By day 3, 30% of B cells isolated from the control mice were dead, whereas only 10% of B cells from AGP-3 transgenic mice were dead. By day 5, 70% of B cells from AGP-3 mice were still viable, whereas only 15% of B cell from control littermates were viable.
  • AGP-3 is a potent B cell stimulatory factor.
  • the increased B cell survival and proliferation may together contribute to the B cell hyperplasia and autoimmune lupus like changes in the AGP-3 transgenic mice.
  • Table 1 Lupus associated autoantibodies in the serum of AGP-3 transgenic mice.
  • ⁇ includes two weak positive. * Weak positive a: Data is shown as number of ANA positive (mean+2sd of transgene negative littermates) mice using ANA screen kit. b: Data is represented as mean+SE for each group. Values are shown as Units/ml. NS: not significant Bacterial Expression of AGP-3 protein
  • PCR amplification employing the primer pairs and templates described below are used to generate various forms of human AGP3 proteins.
  • One primer of each pair introduces a TAA stop codon and a unique Xhol or SacII site following the carboxy terminus of the gene.
  • the other primer of each pair introduces a unique Ndel site, a N-terminal methionine, and optimized codons for the amino terminal portion of the gene.
  • PCR and thermocycling is performed using standard recombinant DNA methodology.
  • the PCR products are purified, restriction digested, and inserted into the unique Ndel and Xhol or SacII sites of vector pAMG21 (ATCC accession no. 98113) and transformed into the prototrophic E. coli 393 or 2596.
  • This construct was engineered to be C-terminal 158 amino acids of human AGP3 and have the following N-terminal and C-terminal residues: NH j -MNSRNKR -GALKLL-COOH.
  • SEQ ID NO: 35 The template to be used for PCR was human AGP3 in pCDNA3.1(+) vector. Oligonucleotides #1761-31 and #1761-33 were the primer pair to be used for PCR and cloning this gene construct.
  • This construct was engineered to be C-terminal 158 amino acids of human AGP3 preceded with FLAG epitope.
  • the template to be used for PCR was human AGP3 in pCDNA3.1(+) vector. Oligonucleotides #1761-32 and #1761-33 were the primer pair to be used for PCR and cloning this gene construct.
  • E. coli were induced during fermentation, the lysate was applied to Q Sepharose FF (Pharmacia, Piscaataway, NJ) equilibrated in 10 mM Mes pH 6.0 and eluted with 50- 400 mM NaCl gradient over 30 column volumes.
  • Q Sepharose FF Puracia, Piscaataway, NJ
  • Fractions containing AGP-3 were pooled and loaded onto a Q Sepharose HP column (Pharmacia, Piscataway, NJ) equilibrated in 10 mM Tris-HCL pH 8.5.
  • AGP-3 was eluted with an increasing linear NaCl gradient (50 mM-200 mM) over 30 column volumes.
  • Endotoxin was removed by application to Sp HiTRAP column (Pharmacia, Piscataway, NJ) pH 4.8 and eluted with 100-500 mM NaCl in 10 mM sodium acetate pH 4.8 over 25 column volumes.
  • Final endotoxin level of the purified protein is approximately 0.2 EU/mg.
  • the purified human AGP-3 is truncated at residue Argl33 as indicated by N-terminal sequencing and has a molecular weight of 16.5 KDa by reducing SDS-PAGE.
  • the purified human FLAG- AGP-3 protein is confirmed by N-terminal sequence analysis of the protein.
  • the FLAG-AGP3 protein is recognized by M2 monoclonal antibody against FLAG epitope (Kodak, New Haven, CT).

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Abstract

L'invention concerne un élément du facteur de nécrose des tumeurs (TNF), les anticorps associés, et leurs utilisations. Cet élément est d'abord exprimé dans les lymphocytes B, et son expression permet de mettre en corrélation l'augmentation du nombre de lymphocytes B et les immunoglobulines produites. L'orthologue humain contient 285 acides aminés, l'orthologue de la souris comprenant 309 acides aminés. La protéine est une protéine transmembranaire de type II, et possède un domaine cytoplasmique aminé terminal, un domaine transmembranaire, et un domaine extracellulaire carboxy terminal. Les protéines de l'invention associées à TNF peuvent être associées à une membrane ou sous forme soluble, produites par recombinaison ou isolées après leur production naturelle. L'invention concerne également des acides nucléiques codant pour les protéines associées à TNF, des vecteurs et des cellules hôtes exprimant les polypeptides, et des techniques de production de protéines de recombinaison. L'invention concerne, en outre, des anticorps, des fragments, des protéines de fusion associées et des dérivés pouvant être utilisés comme agonistes ou antagonistes de l'activité associée à AGP-3.
PCT/US2000/003653 1999-02-12 2000-02-11 Proteines associees au facteur de necrose des tumeurs WO2000047740A2 (fr)

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