JP2001510323A - Secreted protein - Google Patents

Abstract

  (57) [Summary] A new protein is disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION                                 Secreted protein                                Field of the invention   The present invention provides novel proteins and the therapeutic, diagnostic and research utility of these proteins. Related.                                Background of the Invention   Protein factors such as lymphokines, interferons, CSFs and Methods involving the discovery of cytokines such as terleukin) are described in It has matured rapidly in 0 years. Today's routine hybridization clonin Cloning and expression cloning methods provide information directly related to the discovered protein (ie, In the case of hybridization cloning, the partial DNA / amino acid of the protein Sequence; depending on the activity of the protein in the case of expression cloning) Cloning the new polypeptide "directly". Signal sequence cloning (Based on the presence of the currently well-recognized secretory leader sequence motif, Columns), as well as hive by various PCR or with low stringency More recent "indirect" cloning, such as the redidation cloning method This method is secreted in the case of cloning of the leader sequence. Alternatively, depending on the cell or tissue source, the activity may be Make available a large number of DNA / amino acid sequences for known proteins This has raised the current technical level. The present invention relates to these proteins. is there.                                Summary of the Invention   In one embodiment, the present invention provides a polynucleotide selected from the group consisting of: Provided is a composition comprising an isolated protein encoded by a nucleotide:   (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;   (B) nucleotides from nucleotide 230 of the nucleotide sequence of SEQ ID NO: 1 A polynucleotide comprising up to 791;   (C) nucleotide 311 of the nucleotide sequence of SEQ ID NO: 1 A polynucleotide comprising up to 791;   (D) All of clone AM931 deposited under accession number ATCC 98026 A polynucleotide comprising a nucleotide sequence of a long protein coding sequence;   (E) c of clone AM931 deposited under accession number ATCC 98026 Polynucleotide encoding full-length protein encoded by DNA insert Do;   (F) Synthesis of clone AM931 deposited under accession number ATCC 98026 A polynucleotide comprising the nucleotide sequence of a mature protein coding sequence;   (G) c of clone AM931 deposited under accession number ATCC 98026 Polynucleotide encoding mature protein encoded by DNA insert Do;   (H) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 Tide;   (I) Including a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity A polynucleotide encoding a protein;   (J) Polynucleic acid which is an allelic variant of the polynucleotide of (a) to (g) above Nucleotides; and   (K) a polynucleotide encoding a species homolog of the protein of (h) or (i) above Otide.   Preferably, such a polynucleotide is of the nucleotide sequence SEQ ID NO: 1. Nucleotides 230 to 791; nucleotides of SEQ ID NO: 1 Accession number ATCC98 from nucleotide 311 to nucleotide 791 of the sequence No. 026 of full length protein coding sequence of clone AM931 deposited as No. 026 Reotide sequence; or clone A deposited under accession number ATCC 98026 M931 contains the nucleotide sequence of the mature protein coding sequence. Other preferred In a specific example, the polynucleotide is deposited under accession number ATCC 98026. Full length encoded by the cDNA insert of cloned AM931 or Encodes a mature protein. In still other preferred embodiments, such polynucleotides Otide comprises the amino acid sequence from amino acid 32 to amino acid 51 of SEQ ID NO: 2. Encodes a protein.   In another embodiment, the present invention relates to an amino acid selected from the group consisting of: A set comprising a protein comprising an acid sequence and substantially free of other mammalian proteins Serve the product:   (A) the amino acid sequence of SEQ ID NO: 2;   (B) an amino acid sequence from amino acid 32 to amino acid 51 of SEQ ID NO: 2;   (C) fragments of the amino acid sequence of SEQ ID NO: 2; and   (D) c of clone AM931 deposited under accession number ATCC 98026 Amino acid sequence encoded by the DNA insert.   Preferably, such a protein is the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 From amino acid 32 to amino acid 51.   The protein composition of the present invention may further contain a pharmaceutically acceptable carrier. Heel Also provided by the present invention is a composition comprising an antibody that specifically reacts with the protein.   A therapeutically effective amount of a composition comprising the protein of the present invention and a pharmaceutically acceptable carrier, For preventing, treating or ameliorating a medical condition, comprising administering to a subject Is also provided. In the treatment method provided by the present invention, preferably, a vascular form is used. Inhibition of growth, inhibition of vascular endothelial cell growth or proliferation, inhibition of tumor growth and vasculature The subject is treated to produce an effect selected from the group consisting of inhibiting growth-dependent tissue growth. Be treated.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the pED6 and pNotS vectors used for depositing the clones disclosed herein. FIG.Detailed description Isolated protein   Currently determined nucleotide and amino acid sequences are disclosed in The clones and proteins used are shown below. In some examples, the array is Provisional, containing some incorrect or unclear bases or amino acids May be. By performing sequencing of the deposited clones by known methods, The actual nucleotide sequence of the clone can be easily determined. Next, Determine constant amino acid sequence (both full length and mature) from such nucleotide sequence can do. Express the clone in the appropriate cells, collect the protein, and then By determining its sequence, the amino acids of the proteins encoded by the individual clones can be determined. The no acid sequence can also be determined.   For each disclosed protein, applicants must make the most use of the sequence information available at the time of filing. Those that were determined to be well identifiable reading frames were identified. The array being reported The reported protein sequence is "Xaa" because some information is unclear. Including These “Xaas” are (1) unclear nucleotide sequences. (2) (the nucleotide sequence is clearly determined (If any) in the determined nucleotide sequence that the applicant believes cannot be present. Indicates codon.   As used herein, the term "secreted" protein refers to a membrane when expressed in a suitable host cell. Refers to the signal that is transported through Transportation is also included. A “secreted” protein is a protein that is totally secreted from the cell in which it is expressed. White (eg, soluble protein) or partially secreted protein (eg, receptor) Including but not limited to: “Secreted” proteins are also transported across the ER membrane. Includes, but is not limited to, what is sent.   Protein "AM931"   One protein of the present invention was identified as protein "AM931". AM931 Partial cDNAc clone to be selected for cDNA encoding secreted protein Was first isolated from a human fetal kidney cDNA library using conventional methods. Heel The nucleotide sequence of the partial cDNA is determined and the BLASTA / BLASTX and FASTA search The program was searched against the GenBank database. I.M.A.G by search `` Yh63e02.r1 Homo sapiens cDNA clone 13 reported by .E. Consortium No. 4426 5 'to EST (GenBank accession number R32076). At least some identity has been revealed. Also, GenBank contracted by search A hit at number N30331 was also found. The EST database entry The human cDNA clone corresponding to the tree was transferred to the I.M.A.G.E.Consortium library Genome Systems, Inc., St. I ordered from Louis, MO. From the supplier The clones received were tested and full length clones containing the 5 'end and 3' UTR ( (Including a poly A tail). This full-length clone is described herein. It is also called "AM931".   AM931 was identified as encoding a secreted protein by Applicants' method. Was.   The currently determined nucleotide sequence of AM931 is shown in SEQ ID NO: 1. Up The correct reading frame and estimate of the full-length AM931 protein corresponding to the nucleotide sequence. The constant amino acid sequence is shown in SEQ ID NO: 2, which applicant is convinced to be correct. I do. Amino acids 1-27 are a putative leader / signal sequence, The amino acid sequence starts at amino acid 28.   Depositing clones   Clone AM931 was transferred to American Type Culture Coll. On April 17, 1996. , deposited under Accession No. ATCC 98026, and Each containing clone can be obtained from the deposit. Each clone is deposited with this complex Were transfected into separate bacterial cells (E. coli).   EcoRI / NotI digestion (EcoRI at 5 'site, NotI at 3' site) Deposit each clone by performing and obtaining fragments suitable for such clones Can be taken from the vector. Each clone was replaced with pED6 or pNotS in FIG. Deposited in one of the vectors. Expression of cDNA from deposited vector Is also good.   Bacterial cells containing a particular clone can be obtained from a composite deposit as follows. it can:   Oligonucleotide probes to obtain sequences known as specific clones Or a probe should be designed. This sequence is the sequence described herein, or It can be derived from a combination of these sequences.   Preferably, the design of the oligonucleotide probe follows these parameters. Should be:   (A) The region of the sequence where the number of unclear bases (N), if any, is minimal Should be designed to:   (B) Tm of about 80 ° C. (2 ° C. for A or T, G or C, respectively) About 4 ° C.).   Preferably, a method widely used for oligonucleotide labeling is used, Oligonucleotides are converted to γ-³²P-ATP (specific activity 6000 Ci / mmole). Other labeling methods may be used it can. Preferably, the unincorporated label is gel filtered or otherwise established. Should be removed by any method The amount of radioactivity incorporated into the probe It should be quantified by measuring with a titration counter. Preferably, The specific activity of the probe obtained should be about 4e + 6 dpm / pmole.   Preferably, a bacterial culture containing a pool of full-length clones is thawed and 100 μl of Stock was added to 25 ml of sterile L-broth containing 100 μg / ml ampicillin Should be used to inoculate sterile culture flasks containing. Preferably, the culture is It should be grown at 37 ° C. to saturation, and preferably, the saturated culture should be fresh L Should be diluted with broth. Preferably, some of these dilutions are plated 100 μg / ml ampicillin in a 150 mm Petri dish and Grow overnight at 37 ° C. on solid bacterial medium with L-broth containing 5% agar Dilution yields 5000 distinct and well-separated colonies. And volume should be determined. To get clear, well-separated colonies Other known methods can also be used.   The colonies are then nitted using standard colony hybridization techniques. Transfer to a low cellulose filter for dissolution, denaturation and baking. You.   Then, preferably, 0.5% SDS, 100 μg / ml yeast RNA, and And 10 mM EDTA (20X stock is 175.3 gN aCl / liter, 88.2 g sodium citrate, pH 7.0 with NaOH ) (About 10 ml per 150 mm filter) with gentle stirring Incubate at 65 ° C for 1 hour. The probe is then preferably hived. Add to the redidation mix and make the concentration more than 1e + 6dpm / ml Make it equal to this. The filter is then preferably stirred at room temperature. Wash without stirring with 500 ml of 2X SSC / 0.5% SDS, preferably the Afterwards, 500 ml of 2X SSC / 0.1% with gentle shaking at room temperature Wash with SDS. 65 ° C, 30 minutes to 1 hour, 0.1X SSC / 0.5% A third wash with SDS is optimal. Then preferably dry the filter And subject it to autoradiography for sufficient time to visualize positives on X-ray film. You. Other known hybridization methods can also be used.   Positive colonies are picked, grown in medium, and then positively added using standard procedures. Isolate the mid DNA. Then, restriction analysis, hybridization analysis or Clones can be verified by DNA sequencing.   A fragment of the protein of the present invention that can exhibit biological activity is also included in the present invention. You. Protein fragments may be linear or, for example, H.U.Saragovi , Et al., Bio / Technology 10, 773-778 (1992) and R.S. McDowell, et al., J. . Amer. Chem. Soc. 114, 9245-9253 (1992) (both references are incorporated herein by reference). Cyclization using known methods such as those described in Good. For many purposes, including increasing the number of valencies at the protein binding site, Such The fragment may be fused to a carrier molecule along with the immunoglobulin. For example, A protein fragment is fused to the Fc portion of an immunoglobulin via a "linker". You may let it. For a bivalent form of the protein, such a fusion could be made to the Fc portion of an IgG molecule. Can be done against Such fusions using other immunoglobulin isotypes May be obtained. For example, a protein-IgM fusion yields a decavalent form of the protein of the invention. Will be.   The invention also provides the full length and mature forms of the disclosed proteins. Full length form of such a protein Is identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. I have. The mature form of such a protein can be prepared in a suitable mammalian cell or other host cell. Of the disclosed full-length polynucleotide (preferably deposited with the ATCC) May be obtained by The sequence of the mature form of the protein is determined from the amino acid sequence of the full-length form. You may.   The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. versus The corresponding gene can be isolated by a known method using the sequence information disclosed herein. Heel The method comprises the step of providing information on the disclosed sequence for identification and / or a suitable genomic library. Or a probe obtained by amplification of a gene from a source of genomic material or Preparation of primers.   When the protein of the present invention is membrane-bound (eg, a receptor), the present invention Soluble forms are also provided. In such a form, the intracellular and transmembrane domains of the protein And remove all or part of the protein so that it is sufficiently secreted from the host where the protein was expressed. To do. Intracellular and transmembrane domains of the protein of the present invention are determined from sequence information. The domain can be identified by a known method for determining the domain.   Species homologs of the disclosed proteins are also provided by the present invention. Suitable sequences from the sequences shown here Create the appropriate probe or primer, then select an appropriate source of nucleic acid from the desired species. Species homologs may be isolated and identified by screening.   The invention also encompasses allelic variants of the disclosed polynucleotides or proteins. . That is, the present invention provides a polynucleotide encoding the polynucleotide disclosed herein. Naturally occurring isolated polynucleotides encoding one or more homologous or related proteins Different shape State.   The isolated polynucleotide encoding the protein of the present invention is described in Kaufman et al., Nucl. eic Acids Res. PMT2 or pED expression vector disclosed in 19, 4485-4490 (1991) Operably linked to an expression control sequence such as Good. Many suitable expression control sequences are known in the art. In this specification "Operably linked" as defined herein refers to the isolated polynucleotide of the present invention and expression control Polynucleotide / Expression control where the sequence is placed in a vector or cell and ligated To be expressed by a host cell transformed (transfected) with the sequence Means that   Many types of cells can serve as suitable host cells for expression of the proteins of the present invention. Mammalian cells include, for example, monkey COS cells, Chinese hamster ovary (CH O) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells Vesicles, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid Cells, cell lines obtained from in vitro culture of primary tissues, primary explants, H eLa cells, mouse cells, BHK, HL-60, U937, HaK or Jurkat Cells.   Alternatively, in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. It is possible to produce proteins. A potentially suitable yeast strain is Saccharomyces ce revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or different Includes yeast strains capable of expressing the seed protein. A potentially suitable bacterial strain is Escherichia capable of expressing E. coli, Bacillus subtilis, Salmonella typhimurium, or heterologous proteins Functional bacterial strains. If the protein is produced in yeast or bacteria, The protein produced in the above step is, for example, phosphorylated or glycosylated at an appropriate site. May need to be modified to obtain a functional protein. Known chemical or enzymatic Such covalent bonding may be performed using a method.   The isolated polynucleotides of the present invention can be used in one or more insect expression vectors to The protein can be obtained by operably linking to an appropriate control sequence and using an insect expression system. Good. Materials and methods for baculovirus / insect cell expression systems are described, for example, in Kit form from Invitrogen, San Diego, California, USA (MaxBac^Rkit) And such methods are well known in the art and are available from SumTners And Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (198 7) (such as those described herein by reference) is there. As used herein, insect cells capable of expressing the polynucleotide of the present invention Has been "transformed".   Culturing transformed host cells under culture conditions suitable for expressing the recombinant protein The protein of the present invention may be produced more. Next, the obtained expressed protein was subjected to gel filtration and filtration. Such cultures using known purification processes, such as ion exchange chromatography (I.e., culture medium or cell extract). Also, protein Purification is performed on an affinity column containing an agent that will bind to the protein; Canavalin A-agarose, Heparinto Yopal^ROr Cibacrom blue 3GA Fallose^ROne or more columns with affinity columns such as Resin such as phenyl ether, butyl ether, or propyl ether One or more steps using hydrophobic interaction chromatography using Alternatively, it includes immunoaffinity chromatography.   Alternatively, the protein of the invention may be expressed in an easily purified form. For example, Lactose binding protein (MBP), glutathione-S-tonspherase (GST) Or expressed as a fusion protein such as a fusion protein with thioredoxin (TRX) You may. Kits for expression and purification of such fusion proteins are available from New En from glan BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen It is commercially available. Tag a protein with an epitope, and then tag such epitope Purification can also be achieved by using directed, specific antibodies. It takes 1 Pitopes ("flags") are commercially available from Kodak (New Haeven, CT).   Finally, a hydrophobic RP-HPLC medium such as a pendant methyl or other aliphatic group is added. One or more reversed-phase high quality liquid chromatography using silica gel with The protein can be further purified using the-(RP-PLC) step. A few Or a substantially homogeneous isolation system using various combinations of all of the above purification steps. Exchange You can also get protein. The protein thus purified is substantially different from other mammalian proteins. And is defined by the present invention as an "isolated protein".   The protein of the present invention may be used as a product of a transgenic animal. Characterized by somatic or germ cells containing the nucleotide sequence Expressed as an ingredient in milk of transgenic cows, goats, pigs, or sheep Is also good.   The protein may be produced by known conventional chemical synthesis. The present invention by synthetic means Methods for constructing proteins are known to those skilled in the art. The synthetically constructed protein sequence is primary Sharing secondary or tertiary structure and / or conformational properties Thus, they may have common biological properties, including protein activity. Yo For the screening of therapeutic compounds and the generation of antibodies for the production of antibodies. Process to replace the biologically active or immunological replacement of the naturally occurring purified protein May be used.   The protein shown in the present specification has an amino acid sequence similar to that of the purified protein. Which are modified naturally or by derivatization Is also good. For example, modification of a peptide or DNA sequence can be accomplished by one of ordinary skill in the art using known methods. More can be done. The desired modification in the protein sequence depends on the choice in the coding sequence. Includes amino acid residue changes, substitutions, exchanges, insertions or deletions. For example, up to one Or more cysteine residues have been deleted or exchanged for another amino acid. The conformation of the child may be changed. Such changes, replacements, exchanges, insertions, etc. Methods for deletion or deletion are well known to those skilled in the art (see, for example, US Pat. No. 8584). Preferably, such changes, substitutions, exchanges, insertions or deletions It retains the desired activity of white.   Expected to retain protein activity in whole or in part, thus screening Or other fragments of the protein sequence that may be useful for immunological or other immunological methods. Derivatives can also be made by those skilled in the art from the disclosure herein. Such modifications are the subject of the present invention. Is believed to be encompassed byApplications and biological activities   AM931 protein is considered to exhibit angiogenesis inhibitory activity (see Examples below). Also AM931 protein inhibits vascular endothelial cell growth and proliferation. As a result, AM 931 protein is effective in inhibiting angiogenesis (ie, formation of the vasculature). This activity Gender may also be useful in treating tumors or other conditions in which angiogenesis is involved U. Inhibition of angiogenesis by AM931 protein may contribute to normal angiogenesis It may also inhibit or prevent the condition.   Further, the protein of the present invention may have one or more of the following uses or biological activities (the present invention). (Including those associated with the assays cited herein). Departure The use or activity described for a protein may be dependent upon the administration or use of such protein. Or the administration or use of a polynucleotide encoding such a protein ( (E.g., a vector suitable for gene therapy or DNA transfer). Can be.Research applications and usefulness   The proteins provided by the present invention are a family of multiple proteins for high-throughput screening. For assays to determine biological activity, including proteins of interest; production of antibodies or other A protein (or its receptor) in a biological fluid Reagents (including labeling reagents) in assays designed to quantitatively determine The corresponding protein is expressed preferentially (constitutively or with tissue differentiation or Is expressed at a particular stage of development or in a disease state) As well as, of course, to isolate the relevant receptor or ligand You may. Bind when a protein binds or potentially binds to another protein Proteins to identify other proteins and / or inhibitors of binding interactions White can be used. Using proteins involved in these binding interactions, Of peptide or small molecular inhibitor or agonist for binding interaction Training can also be performed.   Any or all of these research uses may be commercialized as research products. Can be developed to reagent grade or kit format.   Methods for performing the above applications are well known to those skilled in the art. Open this method References shown are "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spr. ing Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techn. iques ", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987 But not limited to these.Nutritional uses   The protein of the present invention can be used as a nutrient source or additive. Such uses , As a protein or amino acid additive, as a carbon source, as a nitrogen source And use as a carbohydrate source. Take In some cases, the protein or polynucleotide of the present invention may be added as a food for a particular organism. Well or separate, such as in the form of a powder, pill, solution, suspension or capsule It can also be administered as an individual or as a liquid formulation. In the case of microorganisms, The protein or polynucleotide of the present invention can be added to the medium to be cultured.   Cytokines and cell proliferation / differentiation activity   The protein of the present invention has cytokine activity, cell growth activity (induction or inhibition) or cell activity. Can show blast differentiation activity (induction or inhibition) or in certain cell populations To induce the production of other cytokines. Many proteins found to date Sex factors (including all known cytokines) are dependent on one or more factors Showing activity in cell proliferation assays, and thus the assay Serves as a convenient way to confirm activity. The activity of the protein of the present invention is determined in cell lines (32D, DA 2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (pr eBM +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL 2, including but not limited to TF-1, Mo7e and CMK) Confirmed by any of the many conventional cause-dependent cell proliferation assays for You.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Assays for T cell or thymocyte proliferation are described in Current Protocols in I mmunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. She vach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscienc e (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter ter 7, Immunologic studies in Humans); Takai et al. Immunol. 137: 3494 -3500, 1986; Bertagnolli et al., J. Am. Immunol. 145: 1706-1712, 1990; Bertagno lli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Am. Immunol. 152: 1756-1761 , 1994. But not limited thereto.   Cytokine production and / or expansion of spleen cells, lymph node cells or thymocytes Assays for propagation are described in Polyclonal T cell stimulation, Kruisbeek, A.M. and  Shevach, E.M. In Current Protocols in Imunology. J.E.e.a. Coliganeds. V ol l pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurem ent of mouse and human Interferon γ, Schreiber, R.D. In Current Protoco ls in Immunolog. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley  and Sons, Toronto. 1994. But not limited thereto.         Assays for proliferation and differentiation of hematopoietic and lymphogenic cells asurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly , K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J .E.e.a. coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto o. 1991; deVries et al. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S. A. 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6-Norda n, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp . 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl . Acad. Sci. U.S.A. 83: 1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coliganeds. Vol 1 pp. 6.15.1 John Wil ey and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9- Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Pr otocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wile y and Sons, Toronto. 1991. But not limited thereto.   Assays for the response of T cell clones to antigens (particularly proliferation and APC-T cell interaction as well as direct To identify the effects of T cells) is described in Current Protocols in Immunology, Edby J. et al. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. G reene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro a ssays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellu lar receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al ., Proc. Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al., Eur . J. Immun. 11: 405-411, 1981; Takai et al., J. Am. Immunol. 137: 3494-3500, 19 86; Takai et al. Immunol. 140: 508-512, 1988. But not limited thereto.   Immunostimulatory or suppressive activity   Further, the protein of the present invention has an activity in the assay described in the present specification (not limited thereto). ), And may exhibit immunostimulatory or immunosuppressive activity. Protein is In various deficiencies and disorders, including severe immunodeficiency complications (SCID) May be useful, for example, to increase the proliferation of T and / or B lymphocytes In regulating or down-regulating, and N Useful in enhancing the cytolytic activity of K cells and other cell populations There You. These immunodeficiencies can be genetic and can include viral (e.g., , HIV) and bacterial or fungal infections Or may be caused by an autoimmune disease. More details Include infectious diseases caused by viruses, bacteria, fungi or other infectious agents Diseases, such as HIV, hepatitis virus, herpes virus, mycobacteria, Various fungal infections, such as humania, malaria and Candida spp. It can be treated using Of course, in this regard, a boost to the immune system Is indicated, that is, in treating cancer, the protein of the present invention may be used.   Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis, Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guilla in-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes, myasthenia gravis , Graft-versus-host disease and autoimmune inflammatory eye diseases. Of the present invention Such proteins can be used to treat allergic reactions such as asthma or other respiratory disorders and It may be used to treat symptoms. Other conditions for which immunosuppression is desired (eg, asthma (especially Allergic asthma) and related respiratory problems) using the protein of the present invention. You may be treated. Other conditions in which immunosuppression is desired (including, for example, organ transplantation) The treatment may be performed using the inventive protein.   The proteins of the present invention can also be used to enable an immune response in a number of ways. Da Unregulation can block or block an immune response already in progress Or includes interfering with the induction of an immune response. There may be. By suppressing the T cell response, or Inhibits activated T cell function by inducing resistance or both May be. Generally, immunosuppression of T cell responses is an active, non-antigen specific Process, which requires continuous exposure of T cells to the inhibitor. Resistance and Means non-responsive or anergic in T cells, generally antigen-specific In that it persists after exposure to the tolerizing agent has ceased. Operationally, the lack of a T cell response when exposed to a specific antigen in the absence of a resistance agent More resistance may be shown.   One or more antigen functions (eg, B lymphocyte antigen function (eg, B7) Including, but not limited to, down-regulating For example, blocking high levels of lymphokine synthesis by activated T cells Useful in tissue, skin and organ transplantation and graft versus host disease (GVHD) There will be. For example, blocking T cell function reduces tissue destruction in tissue transplantation Should be. Typically, in tissue transplants, graft rejection is Initiated by T cells being recognized as foreign and then breaking the graft A destructive immune response occurs. B7 Lymphocyte Antigen on Immune Cells and Its Native Riga A molecule that inhibits or blocks interaction with another B lymphocyte antigen (eg, , B7-1, B7-3), in the form of a monomer having the activity of Or a single, soluble, monomeric peptide having B7-2 activity) Pre-implant administration is essential for immune cells without transmitting responsive costimulatory signals. May induce binding of the molecule to its ligand. B lymphocytes in this regard Blocking antigen function depends on cytokine synthesis by immune cells such as T cells. It interferes with growth and thus acts as an immunosuppressant. Besides, lack of co-stimulation May be sufficient to cause a loss of T cell response. B lymphocyte antigen blocking test The induction of long-term tolerance by drugs allows repeated administration of these blocking reagents. The need can be avoided. To achieve sufficient immunosuppression or tolerance in the subject May also need to block the function of the B lymphocyte antigen combination No.   Individual blocking in preventing organ transplant rejection or GVHD Evaluate the effectiveness of the reagents using animal models that can predict their effectiveness in humans be able to. Examples of suitable systems that can be used include allografts in rats and allografts. Xenogeneic pancreatic islet cell grafts in mice and Was used to test the immunosuppressive effect of the CTLA41g fusion protein (Lenscho w et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad. Sci. USA, 89: 11102-11105 (1992)). In addition, GVHD mice Mimodel (Paul ed., Fundamental Immunology, Ravan Press, New York, 1989) , Pp. 846-847) using B lymphocytes to progress the disease in vivo. Anti The blocking effect of the original function can be determined.   In addition, blocking antigen function is therapeutically useful for treating autoimmune diseases It can be. Many autoimmune diseases are responsive to self- Inappropriate activity of T cells to promote the production of cytokines and autoantibodies involved in It is the result of sexualization. Interfering with the activation of self-reactive T cells reduces the symptoms of the disease. Less or may be eliminated. Disrupting B lymphocyte antigen receptor: ligand interaction Inhibit T cell activation using administration of a reagent that blocks T cell costimulation Production of autoantibodies or T cell-derived cytokines that can harm and participate in the disease process Can interfere with life. In addition, blocking reagents can provide long-term remission of disease It may induce antigen-specific resistance of autoreactive T cells that may lead. Autoimmune disease The effectiveness of blocking reagents in the prevention or amelioration of Can be determined using a number of well-characterized animal models You. Examples are murine experimental autoimmune encephalitis, MRLlpr / lpr mice or N Systemic deep erythematosus and murine self-immunity in ZB hybrid mice Plague collagen arthritis, diabetes in NOD mice and BB rats, and Experimental rat myasthenia gravis (Paul ed., Fundamental Immunology, Raven Press) , New York, 1989, pp. 840-856).   Antigen function as a means of up-regulating the immune response (preferred Alternatively, up-regulation of B lymphocyte antigen function) is also therapeutically useful. Up-regulation of the immune response may be May be in a form that eliminates the initial immune response. For example, B lymphocyte antigen function Enhancing the immune response by stimulating may be useful in the case of a viral infection. In addition, systemic viral such as influenza, common cold, and encephalitis Disease may be ameliorated by systemic administration of a stimulatory form of B lymphocyte antigen .   Alternatively, T cells are taken from a patient and tagged with ACPs that express a peptide of the invention. Co-stimulate T cells in vitro with added viral antigens, or Is combined with a stimulating form of the soluble peptide of the invention and then activated in vitro Re-introduced T cells into the patient, the anti-virus in infected patients It may enhance the immune response. Another method of promoting an antiviral immune response is Infected cells are taken from the patient and the nucleic acid encoding the protein of the invention described herein is To allow cells to express all or part of the protein on their surface. And then reintroduce the transfected cells into the patient. U. The infected cells then transmit a costimulatory signal to the T cells in vivo. Could thereby activate T cells.   In another application, antigen function (preferably B lymphocyte antigen function) Up-regulation or promotion may be useful in inducing tumor immunity . Transfection with a nucleic acid encoding at least one peptide of the invention Tumor cells (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, cancer Tumor) can be administered to a subject to overcome tumor-specific resistance in the subject. Desired Transfect tumor cells to express a combination of peptides be able to. For example, a tumor cell obtained from a patient is transformed into a peptide having B7-2-like activity. Peptide having only tide or B7-1-like activity and / or B7-3-like activity Expression vector that directs expression in combination with Can be transfected. Transfected tumor cells To the patient to express the peptide on the surface of the transfected cells. Let Alternatively, transfection in vivo using gene therapy methods To target tumor cells.   Existence of the peptide of the present invention having B lymphocyte antigen activity on the surface of tumor cells Provides the necessary co-stimulatory signals for T cells to provide T cell-mediated trafficking. Induces an immune response against the transfected tumor cells. In addition, MHC Tumor cells lacking class I or MHC class II molecules, or sufficient MHC Tumor cells that cannot reexpress class I or MHC class II molecules are I α chain protein and β_TwoMicroglobulin protein or MHC class II α chain or Or all or part of the MHC class II β chain protein (eg, Transfection with the nucleic acid encoding the Class I or class II MHC proteins can be expressed on the cell surface . Pep having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) Expression of the appropriate Class I or Class II HMC in combination with Tide Induces an immune response to transfected tumor cells mediated by I do. If desired, block expression of MHC class II binding proteins, such as invariant chains. A gene encoding an antisense construct that binds to B lymphocyte antigen Co-transfection with DNA encoding the peptide It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Therefore, Induction of an immune response mediated by a T cell in a human subject is indicative of a tumor in the subject It may be sufficient to overcome specific resistance.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Suitable assays for thymocyte or spleen cell cytotoxicity are described in Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Intersci ence (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; C hapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Ac ad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Am. Immunol. 128: 1968-19 74, 1982; Handa et al., J. Mol. Immunol. 135: 1564-1572, 1985; Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Takai et al., J. Am. Immunol. 140: 508-512, 1988 Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann  et al., J. Amer. Immunol. 128: 1968-1974, 1982; Handa et al., J. Am. Immunol. 135: 1 564-1572, 1985; Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Bowmanet al ., J. Virology 61: 1992-1998; Takai et al., J. Am. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Immunol. 153: 3079-3092, 1994. Including but not limited to the assays described in   Updates for T cell-dependent immunoglobulin response and isotype switching Say (especially to modulate the T cell dependent antibody response to a Th1 / Th2 profile Influencing proteins are identified in Maliazewski, J. Immunol. 144: 3028-3033, 1990. Includes, but is not limited to, the assays described, and further enhances B cell function. Say, In vitro antibody production, Mond, J.J. and Brunswick, M. In Current  Protocols in Immunology J.E.Coliganeds.Val 1 pp.3.8.1-3.8.16, John Wiley  and Sons, Tronto 1994.   Mixed lymphocyte reaction (MLR) assays (especially primarily Th1 and CTL Identify the proteins that produce the answer), Current Protocols in Immunology, Edby J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In V itro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologi c studies in Humans); Takai et al. Immunol. 137: 3494-3500,1986; Takai  et al., J. Amer. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Am. Immunol. 1 49: 3778-3783, 1992. Including but not limited to the assays described in   Dendritic cell-dependent assays (especially for dendritic cells that activate untreated T cells) To identify more expressed proteins)       Guery et al. Immunol. 134: 536-544, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 1991; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et al., Journal of Virology 67: 4062-4069 Huang et al., Science 264: 961-965, 1994; Macatonia et al., Journal  of Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al., Journal o f Clinical Investigation 94: 797-807, 1994; and Inaba et al., Journal of E xperimental Medicine 172: 631-640, 1990. Including but not limited to the assays described in   Lymphocyte survival / apoptosis assays (especially apoptosis after superantibody induction) Identify Proteins That Prevent Lysis and that Regulate Lymphocyte Homeostasis ) Is Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of Immunolog. y 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. But not limited thereto.   Early stage T cell commitment and development assays Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 155: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Natl. Acad. Sci. USA 88: 7548-7551, 1991, including but not limited to Absent.   Hematopoietic regulatory activity   The proteins of the present invention are useful in regulating hematopoiesis and are therefore Useful in the treatment of bulb deficiency. Colony forming cells or factor-dependent cell lines Even minimal biological activity in support of has been implicated in regulating hematopoiesis. For example, to support the growth of erythroid progenitor cells alone, or to In combination with, for example, treatment of various anemias, or release Production of erythroid progenitors and / or erythroblasts in combination with radiation therapy / chemotherapy Stimulating viability; proliferation of bone marrow cells such as granulocytes and monocytes / macrophages Support (ie, traditional CSF activity), eg, in combination with chemotherapy In addition, it is useful in preventing subsequent myelosuppression; Breeding, then supporting platelet growth, and thereby thrombocytopenia It is useful for preventing or treating various platelet diseases, and Used instead of or in addition to platelet infusion; and / or hematopoietic stem Cells (mature to all of the above hematopoietic cells, and therefore various stem cell diseases ( Diseases that are usually treated by transplantation, such as aplastic anemia and development It is useful for supporting the growth of the hemoglobinuria And even after radiation / chemotherapy in vivo or ex vivo (ie, Or combined with bone marrow transplantation) or genetically engineered for gene therapy rear It is also useful in the repopulation of the stem cell compartment as a normal cell.   In particular, the activity of the protein of the present invention may be measured by the following method.   Assays suitable for proliferation and differentiation of various hematopoietic cell lines have already been cited .   Assays for embryonic stem cell differentiation, specifically identifying proteins that affect embryonic hematopoiesis ) Is Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Mol ecular and Cellular Biology 13: 473-486,1993; McClanahan et al., Blood81: 29 Includes, but is not limited to, the assays described in 03-2915,1993.   Assays for stem cell survival and differentiation, specifically identifying proteins that regulate lymphoper hematopoiesis ) Is described in Methylcellulose colony forming assays, Freshney, M.G. In Cultur e of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wi ley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sc i. USA 89: 5907-5911, 1992; Primitive hematopoietic colony forming cells w ith high proliferative potential, McNiece, I.K. and Briddell, R.A. In Cult ure of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wil ey-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22 : 353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21 , Wiley-Liss, Inc .., New York, NY. 1994; Long term bone marrow cultures i n the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T .; In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 16 3-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating  cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Fr eshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 19 94. Including but not limited to the assays described in   Tissue proliferation activity   The protein of the present invention may be used to grow or regenerate bone, cartilage, keys, ligaments and / or nerve tissue. And compositions used for wound healing and tissue repair, further including burns, lacerations You And has utility in treating ulcers.   Book that induces cartilage and / or bone growth in an environment where bone is not formed normally Inventive proteins cure healing of fractures and cartilage damage or defects in humans and other animals There are applications in Such formulations using the protein of the present invention include closed fractures and It is useful for reducing open fractures and improving fixation of artificial joints. Bone De novo bone formation induced by plasticizers can be congenital, traumatic or tumor Contributes to the repair of craniofacial deficits induced by radiological resection and further It is also useful in general surgery.   The proteins of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Heel Agents provide an environment to attract osteogenic cells, stimulate the growth of osteogenic cells, Alternatively, it can induce osteogenic cell precursor differentiation. For example, the protein of the present invention And / or mediated by inflammatory or inflammatory processes Block the process of tissue destruction (collagenase activity, osteoclast activity, etc.) This may be useful in the treatment of osteoporosis or osteoarthritis.   Another category of tissue developmental activity that may be attributed to the proteins of the invention is the key. / Ligament formation. Environments where key / ligament-like or other tissues are not formed properly The protein of the present invention that induces the formation of such a tissue in humans is useful in humans and other animals. Applied to healing of key or ligament lacerations, deformations and other key or ligament defects You. Such a formulation using a key / ligament-like tissue-inducing protein may be used for key or ligament-like tissue. To prevent key or ligament fixation to bone or other tissue May be. The de novo key / ligament-like tissue formation induced by the composition of the present invention Sexual, traumatic, or oncological resection-induced craniofacial defects Cosmetic surgery for contributing to the repair and also for attaching or repairing keys or ligaments It is also useful in The composition of the present invention attracts key- or ligament-forming cells Provide an environment for stimulating the proliferation of key- or ligament-forming cells, Induction of precursors of band-forming cells or tissue repair when returned to vivo. Ex vivo can induce the growth of key / ligament cells or progenitors ex vivo. The compositions of the present invention can be used to treat keratitis, carpal tunnel syndrome and other key or ligament defects. Is also useful. The composition of the present invention may comprise a suitable matrix and / or It may include a sequestering agent, such as a well-known carrier.   The protein of the present invention is used for proliferation of nerve cells and regeneration of nerve and brain tissue, , Central and peripheral nervous system diseases and neuropathy and mechanical diseases and Treatment of traumatic diseases (including degeneration, death or trauma to nerve cells or nerve tissue) Can also be useful for treatment. More specifically, peripheral nerve injury, peripheral neuropathy and Diseases of the peripheral nervous system such as neuropathy and localized neuropathy, and Alzheimer's disease , Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager Proteins may be used in the treatment of diseases of the central nervous system, such as the syndrome. By the present invention Additional conditions that may be treated include mechanical disorders such as spinal cord disorders and traumatic disorders And cerebrovascular diseases such as head trauma and stroke. Chemotherapy or other Peripheral neuropathy caused by medical therapy of It is treatable.   The protein of the present invention may also be used for pressure ulcer, ulcer associated with vascular dysfunction, More preferred for non-healing wounds, including (but not limited to) wounds due to trauma It may also be useful for promoting quick closure.   The protein of the present invention includes organs (eg, pancreas, liver, intestine, kidney, skin, endothelium). ), Muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue To promote the development of other tissues or the proliferation of cells that make up such tissues. Activity. Part of the desired effect is to reduce fibrotic scarring. Regeneration of normal tissue. The proteins of the present invention may also exhibit angiogenic activity.   The protein of the present invention is useful for protecting or regenerating intestine, and for fibrosis of lung or liver, Treatment of tissue reperfusion injury and symptoms caused by systemic cytokine damage Can also be useful for treatment.   A protein of the present invention, which promotes or suppresses differentiation of the above-mentioned tissue from precursor tissue or cells; Alternatively, it may be useful for suppressing the growth of the tissue.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Assay for tissue regeneration activity is described in WO 95/16035 (bone, cartilage, key) ; International Publication WO95 / 05846 (nerves, neurons); International Publication WO91 / 07 491 (skin, endothelium), including but not limited to.   Wound healing activity assays are described in Maibach, HI and Rovee, DT, eds., Year Book M edical Publishers, Inc., Chicago (Eaglstein and Mertz, J. Invest. Dermato l 71: 382-384 (modified by 1978)). Not limited to these.   Activin / inhibin activity   Also, the protein of the present invention may exhibit activin- or inhibin-related activity. I Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), Activins are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). You. Therefore, the protein of the present invention may be used alone or as a member of the inhibin α family. In the form of a heterodimer with a female mammal, which reduces the fertility of a female mammal May be useful as an infertility drug based on the ability of inhibin to reduce spermatogenesis in animals You. Administration of sufficient amounts of other inhibins may result in infertility in these mammals. Can be guided. Alternatively, the proteins of the present invention may be homodimers or Is a heterodimer with other protein subunits of the inhibin-β group Based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells And may be useful as therapeutic agents to induce fertility. For example, US Pat. See 98885. In addition, the protein of the present invention is useful for reproduction in sexually immature mammals. It may be useful for enhancement and, as a result, homes such as cattle, sheep and pigs Increased fertility during the life of the animal.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Assays for activin / inhibin activity are described in Vale et al., Endocrinology 91: 5. 62-572,1972; Ling et al., Nature 321: 779-782,1986; Vale et al., Nature 321: 7 76-779, 1986; Mason et al., Nature 318: 659-663, 1985; Forage et al., Proc. Natl Acad. Sci. USA 83: 3091-3095, 1986, including Not limited to these.   Chemotaxis / chemokinesis activity   The protein of the present invention can be used for mammalian cells such as monocytes, neutrophils, T cells, mast cells, and eosinophils. Chemotactic or chemokinetic activity of spheres and / or endothelial cells (eg, Acting as in). Uses chemotaxis and chemokinesis proteins And recruit or attract the desired cell population to the desired site of action. Chemotaxis Alternatively, chemokinesis proteins may be used to treat and localize tissue injuries and other trauma. It is particularly advantageous for treating localized infections. For example, tumors of lymphocytes, monocytes or neutrophils Attraction to tumor or infected site may improve immune response to tumor or infectious agent You.   Certain proteins or peptides have chemotactic activity for specific cell populations. Direct or indirect, if stimulated, the direction or kinetics of such cell populations. Command movement. Preferably, the protein or peptide has a directed movement of the cell. Have the ability to directly stimulate Specific proteins have chemotactic activity on cell populations Is used to determine whether such proteins or peptides have known chemotaxis Can be easily determined.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Assay for chemotaxis activity (an assay to identify proteins that induce or interfere with chemotaxis) Say) describes the ability of proteins to induce cell translocation across the membrane as well as the ability of one cell population to Includes assays that measure the ability of a protein to induce adhesion to other cell populations. Moving Assays suitable for and adhesion are described in Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. G reene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measure ment of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. In vest. 95: 1370-1376, 1995; Lind et al. APMIS 103: 140-146, 1995; Muller et a l Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152: 5860-58 67, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994. But not limited thereto.   Hemostasis and thrombolytic activity   Further, the protein of the present invention may exhibit hemostatic or thrombolytic activity. As a result Thus, such proteins are useful in the treatment of various clotting disorders, including genetic disorders such as hemophilia. Or in the treatment of injury caused by trauma, surgery or other causes. Blood clots and other hemostasis. The protein of the present invention may be used to dissolve or form thrombus. Growth inhibition and the symptoms resulting therefrom (eg, myocardial infarction and central nervous system blood) It may be useful in the treatment and prevention of vascular infarcts (eg, stroke).   The activity of the protein of the invention may be measured, inter alia, by the following method:   Assays for hemostatic and thrombolytic activity Linet et al., J. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Pr ostaglandins 35: 467-474, 1988, including but not limited to No.   Receptor / ligand activity   Further, the protein of the present invention may be a receptor, a receptor ligand or a receptor / ligand interaction. May exhibit activity as inhibitors or agonists. Such receptors and Examples of gand are cytokine receptors and their ligands, receptor kinases and And their ligands, receptor phosphatases and their ligands, cells Receptors involved in cell interactions and their ligands (cell adhesion molecules (SELEC , Integrin and their ligands) and antigen presentation, antigens Receptor / ligand pairs involved in the development of cognitive, cellular and humoral immune responses Inclusive), but not limited to. Receptors and ligands are related receptors Screening of potential peptide or small molecule inhibitors for body / ligand interactions It is also useful for training. The protein of the present invention (receptor and ligand fragments Include, but are not limited to, blocking the receptor / ligand interaction itself. harm It may be useful as an agent.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Suitable assays for receptor-ligand activity are described in Current Protocols in Immunolog. y, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.S trober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7. 28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6864-6868, 1987; Biere r et al. Exp. Med. 168: 1145-1156, 1988; Rosenstein et al., J. Am. Exp. Me d. 169: 149-160 1989; Stoltenborg et al., J. Am. Immunol. Methods 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995. But not limited thereto.   Anti-inflammatory activity   The protein of the present invention can exhibit anti-inflammatory activity. Anti-inflammatory activity is involved in the stimulus response By stimulating cells, cell-cell interaction (eg, attachment) Chemotaxis of cells involved in the inflammatory process by inhibiting or promoting By inhibiting or promoting, by inhibiting or promoting cell extravasation, Alternatively, it stimulates the production of other factors that more directly inhibit or promote the inflammatory response or Is exhibited by suppressing. Symptoms of inflammation using a protein showing such activity ( Includes chronic or acute symptoms, infection-related inflammation (including septic shock, sepsis) Or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, Fatal injury by dotoxin, arthritis, hyperacute rejection mediated by complement, Nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease Disease, Crohn's disease or overproduction of cytokines such as TNF or IL-1 (Including but not limited to diseases resulting from). Departure Myelin protein is used to treat anaphylaxis and hypersensitivity to antigenic substances or materials. Can also be useful for treatment.Tumor inhibitory activity   In addition to the activities described above for immunological treatment or prevention of tumors, the protein of the present invention Can exhibit antitumor activity. Certain proteins directly or indirectly affect tumor growth (eg, (Via ADCC). Certain proteins are found in tumor or progenitor tissue Act to inhibit the formation of tissue necessary to support tumor growth (eg, angiogenesis) Other factors, agents or cell types that inhibit tumor growth Factors, agents or agents that cause the production of tumors or promote tumor growth Alternatively, tumor-inhibiting activity may be exhibited by removing or inhibiting cell types.   Other activities   The proteins of the present invention may exhibit one or more of the following additional activities or effects: : Infections, including but not limited to bacteria, viruses, fungi and other parasites Sex factor killing; height, weight, hair color, eye color, skin or other tissue pigmentation Or the size or form of an organ or body part (eg, breast augmentation or vice versa) Effects on body characteristics (suppression or promotion), including: fat, protein or charcoal consumed Effects of hydrate on digestion; appetite, libido, stress, cognition (including cognitive disorders), depression Behavioral characteristics, including (but not limited to) depressive disorders and violent behavior Effects; providing analgesic or other pain relief; in non-hematopoietic lineages Promoting differentiation and proliferation of embryonic stem cells; hormonal or endocrine activity; Correct enzyme deficiency and treat related disorders; hyperproliferative disorders (eg, such as psoriasis) ) Treatment; immunoglobulin-like activity (eg, the ability to bind antibodies or complement); And such a protein or protein acting as an antigen in a vaccine composition. Ability to generate an immune response to other substances or entities that cross react with white.Administration and dose   The protein of the invention (from any source, recombinant and non-recombinant) Methods, including, but not limited to, those described above) with a pharmaceutically acceptable carrier. They may be used together in a pharmaceutical composition. Such compositions comprise (in addition to the protein and carrier, ), Diluents, fillers, salts, buffers, stabilizers, solubilizers, and It may contain other well-known substances. The term "pharmaceutically acceptable" A non-toxic substance that does not interfere with the effectiveness of the biological activity of the component. The characteristics of the carrier Depends on the route of administration. The pharmaceutical composition of the present invention comprises M-CSF, GM-CSF, TNF , IL-1, 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, IFN, TNF0, TNF1, TNF2, G-CSF, M eg-CSF, thrombopoietin, stem cell factor, and erythropoietin May contain cytokines, lymphokines, or other hematopoietic factors such as . The pharmaceutical composition further enhances the protein activity, or the activity or May contain other agents that supplement its usefulness. Such additional factors and / or Alternatively, an active agent is contained in the pharmaceutical composition to exert a synergistic effect with the protein of the present invention, Alternatively, side effects may be minimized. Conversely, certain cytokines, lymphokines Originated in prescribing other hematopoietic, thrombolytic or antithrombotic factors, or anti-inflammatory drugs Containing light proteins, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors Side effects of offspring or antithrombotic factors or anti-inflammatory agents may be minimized.   The protein of the present invention may be a multimer (for example, a heterodimer or a homodimer) or It may be active by itself or in a complex with other proteins. As a result, The pharmaceutical composition comprises such a multimeric or complexed form of the protein of the present invention. You may.   The pharmaceutical composition of the present invention may be in the form of a complex of the protein of the present invention and a protein or peptide antigen. It may be in a state. Protein and / or peptide antigens can be It will deliver a stimulus signal to the lymphocytes. B lymphocytes have their surface immunity Will respond to antigen through globulin receptors. T lymphocytes are MHC proteins Will respond to the antigen through the T cell receptor (TCR). MHC and host cell class I and class II MHC genes Structurally related proteins, including those that have been loaded, are peptide-antigens against T lymphocytes. It will help to present the original. The antigen component was a purified MHC-peptide complex only. Or with a co-stimulation schedule that can send signals directly to T cells Is done. Alternatively, it binds to surface immunoglobulins and other molecules on B cells The resulting antibodies can also be mixed with the pharmaceutical compositions of the present invention.   The pharmaceutical composition of the present invention may be in the form of liposome, in which the protein of the present invention is contained. And other pharmaceutically acceptable carriers, amphipathic agents such as lipids (mice in aqueous solution). Agglomerate form, such as insoluble monolayer, liquid crystal or lamellar layer) Have been combined. Suitable lipids for liposome formulations are monoglycerides, diglycerides , Sulfatizide, lysolecithin, phospholipids, saponins, bile acids, etc. However, it is not limited to these. The preparation of such liposome formulations is within the level of ordinary skill in the art. And, for example, U.S. Pat. Nos. 4,235,871, 4501728, 4837028, And 4737323, all of which are described herein by reference. Is considered).   As used herein, the term "therapeutically effective amount" refers to a significant patient benefit, e.g., Sufficient pharmaceutical composition or method to show improvement in symptoms of the condition, healing, increased rate of healing It means the total amount of each active ingredient. Used for individual active ingredients administered alone When used, the term refers only to that component. When used in combinations of active ingredients, The total amount of active ingredients that produces a therapeutic effect, regardless of whether U.   In practicing the treatment method of the present invention, a disease to be treated with a therapeutically effective amount of the protein of the present invention. Administered to a mammal having a morphology. According to the method of the present invention, alone or with a cytokine The protein of the invention together with other therapeutic agents such as lymphokines or other hematopoietic factors. It may be administered. One or more cytokines, lymphokines or other When co-administered with hematopoietic factors, the protein of the present invention may be administered with cytokines, lymphokines, and other It may be administered simultaneously or sequentially with blood, thrombolytic or antithrombotic factors. Gradually When administering the next dose, the attending physician will ask for cytokines, lymphokines, other hematopoietic factors, blood Appropriate administration of thrombolytic factor or antithrombotic factor in combination with the protein of the present invention Will determine the order.   The administration of the protein of the present invention in the pharmaceutical composition of the present invention or used in the practice of the present invention can be carried out by various conventional methods. Administration methods, e.g., oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. I can. Intravenous injection into a patient is preferred.   When a therapeutically effective amount of the protein of the present invention is orally administered, the protein of the present invention may be in the form of a tablet or capsule. , Powder, solution or elixir form. When administered in tablet form, The pharmaceutical composition may further comprise a solid carrier such as gelatin or an adjuvant. May be. Tablets, capsules, and powders contain about 5 to 95% of a protein of the present invention, It preferably contains about 25-90% of the protein of the invention. Place to administer in liquid form Oil, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil , Soybean oil, sesame oil, or synthetic fats and oils may be added. Pharmaceutical group in liquid form The composition can be a saline solution, dextrose or other saccharide solution, or glycol (Eg, ethylene glycol, propylene glycol or polyethylene glycol) Recall). Pharmaceutical compositions when administered in liquid form Is about 0.5 to 90% by weight of the protein of the present invention, preferably about 1 to 50% Contains invention proteins.   When a therapeutically effective amount of the protein of the present invention is administered by intravenous, intradermal or subcutaneous injection, The protein of the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution . Of such a parenterally acceptable protein solution having the appropriate pH, isotonicity, and stability. Preparation is within the skill of the art. Preferred medicament for intravenous, intradermal or subcutaneous injection The composition comprises, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, Dextrose, dextrose and sodium chloride solution for injection, lactic acid for injection Should contain Ringer's solution, or other carriers known in the art . The pharmaceutical composition of the present invention may comprise a stabilizer, a preservative, a buffer, an antioxidant, Other additives known to the consumer.   The amount of the protein of the invention in the pharmaceutical composition of the invention depends on the nature and severity of the condition to be treated, As well as the nature of the treatment the patient has already received. Ultimately, your doctor Each patient will determine the amount of the protein of the invention to be treated. First, the doctor in charge Administer an amount of the protein of the invention and observe the patient's response. Until optimal therapeutic effects are obtained Higher doses of the protein of the present invention may be administered and may be used once optimal therapeutic effect is obtained. amount Do not increase further. Various pharmaceutical compositions for carrying out the method of the present invention may About 0.01 μg to about 100 mg of the protein of the present invention (preferably, About 0.1 μg to about 10 mg per 1 kg of body weight, more preferably 1 kg of body weight 0.1 to about 1 mg of the protein of the present invention.   The duration of treatment by intravenous administration using the composition of the present invention depends on the severity of the disease to be treated. And will vary depending on the condition and response of each patient. Each of the proteins of the present invention The duration of application will range from 12 to 24 hours for continuous intravenous administration . Ultimately, the attending physician will determine the stage of treatment by intravenous administration using the pharmaceutical composition of the present invention. Will decide between.   Immunizing an animal with the protein of the present invention to specifically react with the protein of the present invention; And monoclonal antibodies may be obtained. Whole protein or its fragment Such antibodies may be obtained using immunoglobulin as an immunogen. Carboxy peptide immunogen It may further contain a cysteine residue at the end, and can be used for keyhole rimpet hemoglobin. Binds to haptens such as cyanine (KLH). Those who synthesize such peptides The method is known in the art, for example, R.P.Merrifield, J.Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et.al., FEBS Lett. 211, 10 (1987). Monoclonal antibodies that bind to the protein of the present invention are useful for immunodetection of the protein of the present invention. It can be a diagnostic. Neutralizing antibodies that bind to the protein of the present invention And may be associated with abnormal expression of the protein of the present invention. May be useful in the treatment of certain tumors in the treatment of some forms of cancer You. In the case of cancer cells or white blood cells, a neutralizing monoclonal antibody against the protein of the present invention The body is useful for detecting and preventing metastatic spread of cancer cells that can be mediated by the proteins of the present invention It can be.   For compositions of the invention useful for bone, cartilage, key or ligament regeneration, the method of treatment comprises: The composition can be administered locally, systemically, or locally as an implant or device. Administration. When administered, the therapeutic composition used in the present invention comprises Of course, it is a pyrogen-free, physiologically acceptable form. In addition, Alternatively, the composition may be encapsulated or injected as a viscous form to provide bone, cartilage or May be delivered to the site of tissue damage. Local administration is suitable for wound healing and tissue repair I do. Therapeutically useful action other than the protein of the present invention which may be contained in the above composition The agent is, alternatively or additionally, administered simultaneously or sequentially with the composition in the method of the present invention. May be. Preferably, for bone and / or cartilage formation, the composition comprises Delivering the white-containing composition to the site of bone and / or cartilage damage, and developing the bone and cartilage; Provides a structure for living, and optimally contains a matrix that can be absorbed into the body Will have. Such matrices are currently used for other implanted medical devices It may be made from the materials that have been used.   The choice of matrix material depends on its biocompatibility, biodegradability, mechanical properties, cosmetic Based on appearance and interface properties. The appropriate formulation will be determined by the particular application of the composition. U. Possible matrices for the composition are biodegradable, chemically known sulfuric acid Lucium, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglyco It may be oleic acid and polyanhydride. Other available materials are biodegradable, Well-known in nature, for example, bone or skin collagen. further The matrix may contain pure proteins or extracellular matrix components. Other possible matrices include sintered hydroxyapatite, bioglass, aluminum Not biodegradable, such as silicates or other ceramics, It is. The matrix is a combination of materials of the above type, for example polylactic acid and Including hydroxyapatite or collagen and tricalcium phosphate May be. The bioceramics are added to the composition, for example, calcium-alumina. It may be altered in the to-phosphate and processed to produce pore size, particle size, The particle shape and biodegradability may be varied.   Lactic acid in the form of porous particles having a diameter in the range of 150 to 800 microns and A 50:50 (molar weight) copolymer of biglycolic acid is presently preferred. . In some applications, carboxymethylcellulose or autologous Prevent the dissociation of the protein composition from the matrix using a sequestering agent such as a clot And would be useful.   Preferred families of sequestrants are methylcellulose, ethylcellulose, Roxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl Alkylcells including methylcellose and carboxymethylcellulose With cellulosic materials such as Lurose (including hydroxyalkylcellulose) Yes, cationic salts of carboxymethylcellulose (CMC) are most preferred . Other preferred sequestering agents are hyaluronic acid, sodium alginate, poly (ethylene) Glycol), polyoxyethylene oxide, carboxyvinyl polymer and And poly (vinyl alcohol). The amount of sequestrant useful in the present invention depends on the total formulation. It is 0.5 to 20% by weight, preferably 1 to 10% by weight, and this amount is Prevents protein desorption from the polymer matrix and ensures proper handling of the composition In an amount that allows it, the progenitor cells invade the matrix, To give proteins the opportunity to promote the osteogenic activity of carcinoma cells, there is not much Not to be.   In a further composition, the protein of the present invention comprises a bone and / or cartilage defect, wound, Alternatively, it may be mixed with other agents that are beneficial in treating the tissue. These agents are , Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transformed growth factor (TGF-α and TGF-β) and insulin-like growth factor (IGF) Include.   At present, the therapeutic compositions are also valuable for veterinary use. For details, In addition to humans, livestock and thoroughbred horses are treated with the protein of the present invention. Is a desirable patient.   Rules of administration of protein-containing pharmaceutical compositions used for tissue regeneration alter the effect of proteins Factors, such as tissue weight, damage site, and damage desired to be formed. The condition of the tissue received, the size of the wound, the type of tissue required (eg, bone), the patient's year Consider age, gender and diet, severity of infection, duration of administration, and other clinical factors And your doctor will decide. The dose depends on the type of matrix used for reconstitution. And depending on the content of other proteins in the pharmaceutical composition. For example, IGF   Addition of other known growth factors, such as I (insulin-like growth factor I) to the final composition Addition It can affect the dose. Tissue / bone growth and / or repair can be performed, e.g. Progress through routine assessment by morphological survey and tetracycline labeling Can be monitored.   The polynucleotide of the present invention can also be used for gene therapy. Such polynuks Leotide is introduced into cells in vivo or ex vivo and expressed in a mammalian subject. Can be made. Other known methods for introducing nucleic acids into cells or organisms The polynucleotide of the present invention may be administered more (in a viral vector, Or in the form of naked DNA, but is not limited thereto).   Culturing and growing the cells ex vivo in the presence of the protein of the invention, or Produce the desired effect on such cells or produce the desired activity in such cells. May be. The treated cells are then introduced in vivo for therapeutic purposes. can do.                                 Example 1 Inhibition of angiogenesis and endothelial cell proliferation by AM931 protein   The vasostatic (angiogenesis inhibition) activity of AM931 protein was assayed for vasostatic activity. HUVC-endothelial cell proliferation assay. 96 assays Performed on the plate. Primary human umbilical cord cells (HUVECs) were transformed into EGM medium (Clonetics) / 3x10 per well in 20% FCS (fetal calf serum)^ThreeScatter And incubated at 37 ° C. for 24 hours. The cells are then replaced with 0.5% FC Starved at 37 ° C. for 48 hours in S-containing M199 medium (M199-0.5% FCS) State. If obtained from COS-1 cells transfected with AM931 Medium or membrane fraction was washed with M199 containing 100 ng / ml EGF. -Diluted 1: 5, 1:25, 1: 125 and 1: 625 with 0.5% FCS . Add a dilution of AM931 to the starved cells and incubate at 37 ° C for 72 hours. I did. The cells are then [^ThreeRadiolabeled with [H] -thymidine for 6 hours. radiation Wash labeled cells with PBS for liquid scintillation counting, trypsinize Was. Plot results using Kaleidograph software (Abelbeck Software) did.   The effect of various formulations on HUVEC growth is shown in FIGS. 2A and 2B. FIG. In A and 2B, “pED” was transfected with the expression vector only. Mock control from loaded COS-1 cells. "AM931 CM (1)" And “AM931 CM (2)” are two separate COS-1 cell transformants This is a conditioned medium. “AM931 MF” is the second COS-1 transformer It is a membrane fraction obtained by the infection. These results indicate that angiogenesis is Shows the activity of AM931 protein in harm (vasostasis) and inhibition of vascular endothelial proliferation You.   The patents and publications cited herein are hereby incorporated by reference in their entirety. It is assumed that

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/435 A61K 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP, MX (72) Inventor McCoy, John M. Howard Redding, Reading, Massachusetts, USA 01867 Street 56 (72) Inventor Lacey, Lisa A. United States 01720 Acton, Mass., School Street 124 (72) Inventor Lavery, Edward Earl United States 01876 Massachusetts, Tuxbury, Green Street 90 (72) )invention Marburg, David United States 01720 Orton, Massachusetts, Orchard Drive No. 2 (72) Inventor Spalding, Vicky United States 01821 Villarica, Mass., No. 11 Meadowbank Road

Claims (1)

[Claims]   1. (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;   (B) nucleotides from nucleotide 230 of the nucleotide sequence of SEQ ID NO: 1 A polynucleotide comprising up to 791;   (C) nucleotide 311 of the nucleotide sequence of SEQ ID NO: 1 A polynucleotide comprising up to 791;   (D) All of clone AM931 deposited under accession number ATCC 98026 A polynucleotide comprising a nucleotide sequence of a long protein coding sequence;   (E) c of clone AM931 deposited under accession number ATCC 98026 Polynucleotide encoding full-length protein encoded by DNA insert Do;   (F) Synthesis of clone AM931 deposited under accession number ATCC 98026 A polynucleotide comprising the nucleotide sequence of a mature protein coding sequence;   (G) c of clone AM931 deposited under accession number ATCC 98026 Polynucleotide encoding mature protein encoded by DNA insert Do;   (H) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 Tide;   (I) Including a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity A polynucleotide encoding a protein;   (J) Polynucleic acid which is an allelic variant of the polynucleotide of (a) to (g) above Nucleotides; and   (K) a polynucleotide encoding a species homolog of the protein of (h) or (i) above Otide Comprises an isolated protein encoded by a polynucleotide selected from the group consisting of: Composition.   2. The composition of claim 1 further comprising a pharmaceutically acceptable carrier.   3. Administering to a mammalian subject a therapeutically effective amount of the composition of claim 2. How to prevent, treat or ameliorate a medical condition.   4. A protein comprising an amino acid sequence selected from the group consisting of: A composition comprising a protein substantially free of other mammalian proteins: Serve the product:   (A) the amino acid sequence of SEQ ID NO: 2;   (B) an amino acid sequence from amino acid 32 to amino acid 51 of SEQ ID NO: 2;   (C) fragments of the amino acid sequence of SEQ ID NO: 2; and   (D) c of clone AM931 deposited under accession number ATCC 98026 Amino acid sequence encoded by the DNA insert.   5. The composition of claim 4, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2. .   6. The protein is an amino acid sequence from amino acid 42 to amino acid 61 of SEQ ID NO: 2; 5. The composition of claim 4, wherein said composition comprises rows.   7. 3. The composition of claim 2, further comprising a pharmaceutically acceptable carrier.   8. Administering to a mammalian subject a therapeutically effective amount of the composition of claim 7. How to prevent, treat or ameliorate a medical condition.   9. The polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1. The composition of claim 1.   10. The polynucleotide is a nucleotide from nucleotide 230 of SEQ ID NO: 1. The composition of claim 1 which comprises a nucleotide sequence up to tide 791.   11. The polynucleotide is a nucleotide from nucleotide 311 of SEQ ID NO: 1 The composition of claim 1 which comprises a nucleotide sequence up to tide 791.   12. The polynucleotide deposited with accession number ATCC 98026. It contains the nucleotide sequence of the full length protein coding sequence of Lone AM931 The composition of claim 1.   13. The clone in which the nucleotide has been deposited under accession number ATCC 98026. Encoding the full-length protein encoded by the cDNA insert of AM931. 2. The composition of claim 1, wherein said composition comprises a polynucleotide.   14． The polynucleotide deposited with accession number ATCC 98026. Containing the nucleotide sequence of the mature protein coding sequence of Lone AM931 The composition of claim 1.   15. The polynucleotide deposited with accession number ATCC 98026. Encoding the mature protein encoded by the cDNA insert of Lone AM931 2. The composition of claim 1, wherein said composition comprises a polynucleotide.   16. The polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 2 2. The composition of claim 1, wherein said composition comprises a polynucleotide.   17． The protein is an amino acid from amino acid 32 to amino acid 51 of SEQ ID NO: 2; 5. The composition of claim 4, comprising a sequence.   18. Clone AM9 whose protein was deposited under accession number ATCC 98026 A sequence containing the amino acid sequence encoded by the 31 cDNA insert The composition of claim 4.   19. 5. The composition of claim 4, further comprising a pharmaceutically acceptable carrier.   20. Administering to a mammalian subject an effective amount of the composition of claim 19. To prevent, treat or ameliorate a biological condition.   21. Inhibition of angiogenesis, inhibition of vascular endothelial cell growth or proliferation, inhibition of tumor growth Produces an effect selected from the group consisting of harm and inhibition of angiogenesis-dependent tissue growth 9. The method of claim 8, wherein said subject is treated.   22. Inhibition of angiogenesis, inhibition of vascular endothelial cell growth or proliferation, inhibition of tumor growth Produces an effect selected from the group consisting of harm and inhibition of angiogenesis-dependent tissue growth 21. The method of claim 20, wherein said subject is treated.