JP2002516563A - Secreted proteins and polynucleotides encoding them - Google Patents

Abstract

  (57) [Summary] Disclosed are novel polynucleotides and the proteins encoded by them.

Description

DETAILED DESCRIPTION OF THE INVENTION Secreted Proteins and Polynucleotides Encoding The Same The present application claims the name of "secreted proteins and the polynucleotides encoding them" in the name of some or all of the inventors on August 2, 1996. This is a continuation-in-part of Japanese Patent Application No. 08/691641. FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, as well as therapeutic, diagnostic and research utilities for these polynucleotides and proteins. BACKGROUND OF THE INVENTION Methods aimed at the discovery of proteinaceous factors (including, for example, cytokines such as lymphokines, interferons, CSFs and interleukins) have matured rapidly in the last decade. At present, conventional hybridization and expression cloning methods rely on information directly related to the protein discovered (ie, the partial DNA / amino acid sequence of the protein in the case of hybridization cloning; the activity of the protein in the case of expression cloning). )), The new polypeptide is cloned "directly". More recent "indirect" methods, such as signal sequence cloning (isolating DNA sequences based on the presence of the now well-recognized secretory leader sequence motif), as well as various PCR or low stringency hybridization cloning methods. "Cloning methods involve a number of proteins known to have biological activity due to their secreted nature in the case of cloning of the leader sequence, or due to cellular or tissue sources in the case of PCR methods. The state of the art has been advanced by making available DNA / amino acid sequences. The present invention is directed to these proteins and the polynucleotides that encode them. In a specific embodiment of the invention, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 from nucleotide 309 to nucleotide 509; (c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BL15 12 deposited under accession number ATCC 98154; A) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL1512 deposited under accession number ATCC 98154; (e) the mature protein coding of clone BL1512 deposited under accession number ATCC98154 (F) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL1512 deposited under accession number ATCC 98154; (g) a polynucleotide comprising the amino acid sequence of SEQ ID NO: 3. (H) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 3 having biological activity; (i) a polynucleotide encoding a protein comprising (J) a polynucleotide that encodes a species homolog of the protein of (g) or (h) above. Preferably, such a polynucleotide has been deposited as accession number ATCC 98154; nucleotide sequence from nucleotide 309 to nucleotide 509 of SEQ ID NO: 2; nucleotide sequence of the full length protein coding sequence of clone BL1512 deposited as accession number ATCC 98154; Contains the nucleotide sequence of the mature protein coding sequence of clone BL1512. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BL1512 deposited under accession number ATCC 98154. Other embodiments provide a gene corresponding to the cDNA sequence of SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 4. In another embodiment, BL15 12 in any of the above may be replaced with isolate BL151 deposited under accession number AT CC98117. In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Further, a method for producing a protein, comprising: (a) growing a culture of a host cell transformed with such a polynucleotide composition in a suitable medium; and (b) purifying the protein from the culture. There is also provided a method comprising: The protein produced by such a method is also provided by the present invention. Preferred specific examples include those in which the protein produced by such a method is a mature form of the protein. The protein composition of the present invention may further contain a pharmaceutically acceptable carrier. Compositions comprising antibodies specifically reactive with such proteins are also provided by the present invention. Also provided is a method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the invention and a pharmaceutically acceptable carrier. DETAILED DESCRIPTION Isolated Proteins and Polynucleotides The nucleotide and amino acid sequences for each of the clones and proteins disclosed herein are reported below. In some instances, the sequence is provisional and may include some incorrect or undefined bases or amino acids. By sequencing the deposited clones by known methods, the actual nucleotide sequence of each clone can be readily determined. The deduced amino acid sequence (both full length as well as mature) can then be determined from such nucleotide sequences. By expressing the clones in a suitable host cell, collecting the proteins, and then determining the sequence, the amino acid sequence of the protein encoded by each clone can also be determined. For each protein disclosed, Applicants have identified what has been determined to be the best identifiable reading frame using sequence information available at the time of filing. The reported protein sequence contains the symbol "Xaa" because some of the reported sequence information is unclear. These symbols "Xaa" can be (1) residues that cannot be identified because the nucleotide sequence is unclear, or (2) (if the nucleotide sequence was more clearly determined) the applicant could not be present. The stop codon in the determined nucleotide sequence considered to be considered is shown. As used herein, the term "secreted" protein refers to that which is transported across a membrane when expressed in a suitable host cell, including transport as a result of a signal sequence in its amino acid sequence. “Secreted” proteins include, but are not limited to, proteins that are totally secreted (eg, soluble proteins) or partially secreted (eg, receptors) from the cell in which they are expressed. "Secreted" proteins also include, but are not limited to, those that are transported across the endoplasmic reticulum membrane. Clone "BL15 12" The polynucleotide of the present invention was identified as clone "BL15 12". BL1512 was isolated from an adult testis cDNA library using methods selective for cDNAs encoding secreted proteins. BL1512 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BL1512 protein"). The nucleotide sequence of the 5 ′ portion of BL1512 determined this time is shown in SEQ ID NO: 1. The additional internal nucleotide sequence from BL1512 determined this time is shown in SEQ ID NO: 2. What Applicants currently consider to be the correct reading frame and deduced amino acid sequence encoded by such an internal sequence is shown in SEQ ID NO: 3. Additional nucleotide sequence of the 3 'portion of BL1512, including the poly A tail, is shown in SEQ ID NO: 4. The nucleotide sequence disclosed herein for BL1512 was searched against the GenBank database using the BLASTA / BLASTX and FASTA search protocols. No hits were found in the database. Clone Deposit Clone BL1512 has been deposited with the American Type Culture Collection on August 23, 1996 as Accession No. ATCC 98154 as part of a composite deposit having accession number ATCC 98117, from which the clone is available. Each clone was transfected into separate bacterial cells (E. coli) as this complex deposit. An EcoRI / NotI digestion (5 'site, EcoRI; 3' site, NotI) was performed to obtain an appropriately sized fragment (approximately the clone size fragment was identified as follows) for such clones. Can be taken from the deposited vector. Bacterial cells containing a particular clone can be obtained from the composite deposit as follows: The oligonucleotide probe (s) should be designed to have a known sequence for that particular clone. It is. This sequence can be derived from the sequences herein or a combination of those sequences. The sequence of the oligonucleotide probe used to isolate each full-length clone was identified as follows and should be the most reliable in isolating the clone of interest. Clone probe sequence BL15 12 SEQ ID NO: 5 In the above sequence containing an N at position 2, that position may be a single nucleotide (eg, biotin phosphoramidite (1-dimethoxytrityloxy-2- ( Using N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanoethyl)-(N, N-diisopropyl) -phosphoramadite (Glen Research, cat. No. 10-1953)) ), Rather than biotinylated phosphoramidite residues. Preferably, the design of the oligonucleotide probe should follow these parameters: (a) Should be designed to be the region of the sequence where the number of undefined bases (N), if any, is minimal; (b) T _m of about 80 ° C. (respectively, 2 ° C. for a or T, for G or C is 4 ° C. assumed) should be designed so as to. Preferably, oligonucleotides should be labeled with g- ³² P-ATP (specific activity 6000 Ci / mmol) using T4 polynucleotide kinase, using methods commonly used for oligonucleotide labeling. Other labeling methods can be used. Preferably, unincorporated label should be removed by gel filtration chromatography or other established methods. It should be quantified by measuring the amount of radioactivity incorporated into the probe with a scintillation counter. Preferably, the specific activity of the resulting probe should be about 4e + 6 dpm / picomolar. Preferably, the bacterial culture containing the pool of full-length clones should be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L broth containing 100 μg / ml ampicillin. Preferably, the culture should be grown to saturation at 37 ° C., and preferably, the saturated culture should be diluted with fresh L-broth. Preferably, a portion of these dilutions are plated out in a 150 mm Petri dish with 100 μg / ml ampicillin and 1. The dilution and volume at which approximately 5000 distinct, well-separated colonies result when grown overnight at 37 ° C. on solid bacterial culture medium containing L broth containing 5% agar should be determined. is there. Other known methods for obtaining well-defined and well-separated colonies can also be used. The colonies should then be transferred to nitrocellulose filters using standard colony hybridization techniques, lysed, denatured, and then baked. Then, preferably 6X SSC containing 0.5% SDS, 100 μg / ml yeast RNA and 10 mM EDTA (20X stock is 175.3 g NaCl / liter, 88.2 g sodium citrate / liter, pH 7.0 with NaOH). Incubate the filter at 65 ° C. for 1 hour with gentle agitation in (approximately 10 ml per 150 mm filter). The probe is then preferably added to the hybridization mixture so that the concentration is greater than 1e + 6 dpm / ml. It is then preferred to incubate overnight at 65 ° C. with gentle agitation. The filter is then preferably washed with 500 ml 2X SSC / 0.5% SDS without stirring at room temperature, preferably followed by 15 minutes with 500 ml 2X SSC / 0.1% SDS at room temperature with gentle shaking. Wash for minutes. The third wash is performed at 65 ° C. for 30 minutes to 1 hour, at 0. Optimally, it is performed with 1X SSC / 0.5% SDS. The filters are then preferably dried and subjected to autoradiography for a sufficient time to visualize positives on X-ray film. Other known hybridization methods can be used. Positive colonies are picked, grown in medium, and the plasmid DNA is then isolated using standard procedures. The clone can then be verified by restriction analysis, hybridization analysis or DNA sequencing. A fragment of the protein of the present invention that can exhibit biological activity is also included in the present invention. Protein fragments may be linear or, for example, HUSaragovi, et al., Bio / Technology 10, 773-778 (1992) and RSM C Dowell, et al., J. Am. Amer. Chem. Soc. Cyclization may be performed using known methods as described in 114, 9245-9253 (1992) (both references are deemed to be described herein by reference). Such fragments may be fused to a carrier molecule, such as an immunoglobulin, for a number of purposes, including increasing the number of valencies at the protein binding site. For example, a protein fragment may be fused to the Fc portion of an immunoglobulin via a "linker" sequence. For bivalent forms of the protein, such fusions can be made to the Fc portion of an IgG molecule. Such fusions may be performed using other immunoglobulin isotypes. For example, a protein-IgM fusion will produce a ten-valent form of a protein of the invention. The invention also provides the full length and mature forms of the disclosed proteins. The full length form of such a protein has been identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. Mature forms of such proteins may be obtained by expressing the disclosed full-length polynucleotides (preferably those deposited with the ATCC) in appropriate mammalian cells or other host cells. The sequence of the mature form of the protein may be determined from the amino acid sequence of the full-length form. The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. Corresponding genes can be isolated by known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information for identification and / or amplification of genes in a suitable genomic library or other source of genomic material, and / or Includes amplification of genes in rally or other sources of genomic material. Where a protein of the invention is membrane-bound (eg, if it is a receptor), the invention also provides a soluble form of such protein. In such a form, some or all of the intracellular and transmembrane domains of the protein are deleted so that the protein is sufficiently secreted from the cell in which it was expressed. The intracellular and transmembrane domains of the proteins of the present invention can be identified by known techniques for determining such domains from sequence information. Species homologs of the disclosed proteins are also provided by the present invention. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein and screening for an appropriate nucleic acid source from the desired species. The invention also includes allelic variants of the disclosed polynucleotides or proteins. That is, the present invention encompasses naturally occurring alternative forms of the isolated polynucleotide that also encode the same, homologous or related protein as the protein encoded by the polynucleotide. The isolated polynucleotide of the present invention can be prepared as described in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), the protein may be recombinantly produced by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed. Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known and are described, for example, in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). “Operably linked,” as defined herein, refers to a polynucleotide or expression control sequence that is placed in a vector or cell, wherein the isolated polynucleotide and expression control sequence of the invention are transformed with the protein / ligated polynucleotide / expression control sequence. Transfected) means to be expressed by the transfected host cell. Many types of cells can serve as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, and other transformed cells. Includes primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. I do. Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. Potentially suitable yeast strains include Saccharomyces ce revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or yeast strains capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escheric hia coli, Bacillus subtilis, Salmonella typhimurium, or bacterial strains capable of expressing heterologous proteins. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced therein, for example, by phosphorylation or glycosylation at appropriate sites to obtain a functional protein. Such covalent linkages may be made using known chemical or enzymatic methods. The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors, and the protein obtained by using an insect expression system. Materials and methods for baculovirus / insect cell expression systems, e.g. Invitrogen, San Diego, California, are commercially available in kit form from USA (MaxBac ^R kit), such methods are well known in the art , Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (11987), which is hereby incorporated by reference. As used herein, an insect cell capable of expressing the polynucleotide of the present invention has been “transformed”. The protein of the present invention may be produced by culturing the transformed host cell under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein may then be purified from such culture (ie, culture medium or cell extract) using known purification processes such as gel filtration and ion exchange chromatography. Purification of the protein may also be achieved by affinity columns containing an agent that will bind to the protein; one or more column columns with an affinity column such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibacrom blue 3GA Sepharose ^R ; One or more steps using hydrophobic interaction chromatography with a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may be expressed in an easily purified form. For example, it may be expressed as a fusion protein such as a fusion protein with maltose binding protein (MBP), glutathione-S-tonsferase (GST) or thioredoxin (TRX). Kits for the expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and then purified by using a specific antibody directed against such epitope. One such epitope ("flag") is commercially available from Kodak (New Haeven, CT). Finally, the protein is further purified using one or more reverse phase high quality liquid chromatography (RP-PLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. be able to. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined by the present invention as an "isolated protein". The protein of the present invention is expressed as a product of a transgenic animal, for example, as a component of milk of a transgenic cow, goat, pig, or sheep characterized by somatic cells or germ cells containing a nucleotide sequence encoding the protein. You may. The protein may be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed protein sequences may share common biological properties, including protein activity, by sharing primary, secondary, or tertiary structure and / or conformational properties. . Thus, they may be used as biological activities or immunological replacements for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and for generating antibodies. The proteins described herein are characterized by an amino acid sequence similar to the amino acid sequence of the purified protein, but may be modified therein, either naturally or by derivatization. For example, modifications of the peptide or DNA sequence can be made by those skilled in the art using known methods. Modifications of interest in the protein sequence include changes, substitutions, exchanges, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Methods for such alterations, substitutions, exchanges, insertions or deletions are well known to those skilled in the art (see, for example, US Pat. No. 4,158,584). Preferably, such changes, substitutions, exchanges, insertions or deletions retain the desired activity of the protein. Other fragments and derivatives of the protein sequence that are expected to retain protein activity in whole or in part, and thus may be useful in screening or other immunological methods, can also be made by those skilled in the art from the disclosure herein. . Such modifications are believed to be encompassed by the present invention. Uses and Biological Activities The polynucleotides and proteins of the present invention are believed to exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, such as a vector suitable for gene therapy or DNA transfer). Can be provided. Research Uses and Utility The polynucleotides provided by the present invention can be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes "Gene chips" or other probes as probes for subtraction To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization techniques, and to generate anti-DNA antibodies or to generate additional immunizations. Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Can be used in an interaction trap assay (eg, as described in Gyuris et al., Cell 75: 791-803 (1993)). Assays for determining biological activity, including the proteins provided by the present invention, and a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively). Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker of tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. References disclosing such methods are found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, Sambrook, J., EF. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, SL. and AR. Including, but not limited to, Kimmel eds., 1987. Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate individual or liquid formulation, such as in the form of a powder, pill, solution, suspension or capsule. it can. In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured. Cytokines and Cell Proliferation / Differentiation Activity The proteins of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or induce the production of other cytokines in certain cell populations. sell. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention is determined by the cell lines (32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (prEBM +), 2E8, RB5, DA1, 123, T1165, HT2, (Including, but not limited to, CTL L2, TF-1, Mo7e, and CMK). 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 Immunology, Ed. E. Coligan, AM. Kruisbeek, DH. Margulies, EM. She vach, W Strober, Pub. J. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitroassays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al. Immunol. 137: 3494--3500, 1986; Bertagnolli et al., J. Am. Immunol. 145: 1706-1712, 1990; Bertagnol li et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., J. Am. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Am. Immunol. 152: 1756-1761, 1994, but is not limited thereto. Assays for cytokine production and / or proliferation of spleen cells, limb node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM. and Shevach, EM. In Current Protocols in Imlunology. JEea. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, RD. In Current Protocols in Immunology. JEea. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. Includes, but is not limited to, those described in 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, LS. and Lipsky, PE. In Curent Protocols in Immunology. JEea. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 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. USA. 80:29 31-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R.M. In Current Protocols in Immunology. JEea. Coligan eds. Vol 1 pp. 6.6.1- 6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. USA. 83: 1857-1861, 1986; Measurement of human Interleukin 11-Ben nett, F., Giannotti, J., Clark, SC. and Turner, K. J. In Current Protocols in Immunology. JEea. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991: Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC. and Turner, KJ. In Current Protocols in Immunology. JEea. Coligan eds. Vol 1 pp. 6.13.1, Jolm Wiley and Sons, Toronto. Includes, but is not limited to, those described in 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions and direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Edby J. et al. E. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitroas says for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors: Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al., Eur. J. Immum. 11: 405-411, 1981; Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Takai et al., J. Am. Immunol. 140: 508-5 12, 1988, including, but not limited to. Immunostimulatory or Suppressive Activity The protein of the invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to the activity in the assays described herein. Proteins may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), for example, in up-regulating or down-regulating T and / or B lymphocyte proliferation. , And in activating the cytolytic activity of NK cells and other cell populations. These immunodeficiencies may be genetic and may be caused by viruses (eg, HIV) and bacterial or fungal infections, or may be caused by autoimmune diseases . More specifically, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, for example, various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated using the protein of the present invention. Of course, in this regard, the proteins of the present invention may be used where a boost to the immune system is indicated, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis, systemic deep erythroides, rheumatoid arthritis, autoimmune pneumonia, Guilla in-Barre syndrome, autoimmune thyroid Includes inflammation, insulin-dependent diabetes, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions in which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems, may be treated with the proteins of the invention. Other conditions in which immunosuppression is desired, including, for example, organ transplantation, may be treated with the proteins of the present invention. The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response already in progress, or may involve interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped. Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (eg, such as B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the graft is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Pre-implant administration of a soluble, monomeric form of the peptide with B7-2 activity, mixed with the peptide alone or alone, can result in the binding of the native ligand on immune cells to the immune cell without transmitting a responsive costimulatory signal. May induce molecular binding. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Described in Lenschow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad. Sci. USA, 89: 11102-11105 (1992)). Further, using a murine model of GVHD (see Pauled., Fundamental Immunology, Ravan Press, New York, 1989, pp. 846-847), the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo was examined. Can be determined. Also, blocking antigen function can be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples are murine experimental autoimmune encephalitis, systemic deep erythematosus in MRL lpr / lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and experimental murine myasthenia in rats. Disease (Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful. Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection. In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of stimulatory forms of B lymphocyte antigen. Alternatively, the T cells are taken from a patient and co-stimulated in vitro with the viral antigen to which the ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of the soluble peptide of the invention and then in vitro. By reintroducing the T cells activated in the above into the patient, the antiviral immune response in the infected patient may be promoted. Another method of promoting an antiviral immune response is to take infected cells from a patient and transfect them with a nucleic acid encoding a protein of the invention described herein so that the cells have all or part of the protein on their surface. Will be expressed and then the transfected cells will be reintroduced into the patient. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with a peptide having B7-2-like activity alone or with a peptide having B7-1-like activity and / or B7-3-like activity, It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, tumor cells can be targeted for transfection in vivo using gene therapy. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, tumor cells lacking MHC class I or MHC class II molecules, or tumor cells that cannot re-express a sufficient amount of MHC class I or MHC class II molecules, are transformed into MHC class I α chain protein and β ₂ microglobulin protein or MHC class II α Transfected with nucleic acids encoding all or part of the MHC class II β-chain protein (eg, the cytoplasmic domain of the truncated protein), thereby expressing the class I or class II MHC protein on the cell surface be able to. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is a T cell-mediated transfected tumor Induces an immune response against the cells. If desired, a gene encoding an antisense construct that blocks expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject. The activity of the protein of the invention may be measured, inter alia, by the following method: An assay suitable for thymocyte or spleen cell cytotoxicity is described in Current Protocols in Immunology, Ed. E. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitroassays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Am. Immunol. 128: 196 8-1974, 1982; Handa et al., J. Am. Immunol. 135: 1564-1572, 1985; Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Takai et al., J. Am. Immu nol. 140: 508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:24 88-2492, 1981; Herrmann et al., J. Am. Immunol. 128: 1968-1974, 1982; Handa et al., J. Am. Immunol. 135: 1564-1572, 1985: Takai et al., J. Am. Immunol. 137: 3494--3500, 1986: Bowman et al., J. Am. Virology 61: 1992-1998; Takai et al., J. Am. Immun unol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Am. Immunol. 153: 3079-3092, 1994, including, but not limited to. Assays for T cell-dependent immunoglobulin responses and isotype switching, specifically to modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described in Maliazewski, J. Immunol. 144: 3028-3033, 1990, including but not limited to assays of B cell function, as described in In vitro anti body production, Mond, JJand Brunswick, M. In Current Protocols in Immunology JEColigan eds. Val 1 pp. 3.8.1-3.8.16, John Wyley and Sons, Toronto 1994. Mixed lymphocyte reaction (MLR) assays, which identify proteins that primarily produce Th1 and CTL responses, are described in Current Protocols in Immunology, Ed. E. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunological studies in Humans); Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Takai et al., J. Am. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Am. Immunol. 149: 3778-3783, 1992, but is not limited thereto. Dendritic cell-dependent assays (particularly identifying proteins expressed by dendritic cells that activate intact T cells) are described by Guery et al., J. Am. 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, 1993; Huang et al., Science 264: 961-965, 1 994; Macatonia et al., Journal of Experimental Medicine 169. : 1255-1264, 1989; including the assays described in Bhardwaj et al., Journal of Clinical Investigation 94: 797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172: 631-640, 1990. But not limited to them. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by 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; Itohetal., Cell 66: 233-243, 1991; Zacharchuk, Journal of Immunology 145: 4037-. 4045, 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., International Journal of Oncology 1: 639-648, 1992, including but not limited to. Early stage T cell commitment and developmental assays are described in 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. Hematopoietic modulatory activity The proteins of the present invention are useful in regulating hematopoiesis and therefore in treating bone marrow and lymphocyte deficiencies. Even minimal biological activity supporting colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis. For example, supporting the proliferation of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing usefulness in the treatment of various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all hematopoietic cells and therefore supports the growth of a variety of stem cell diseases, which are usually treated by transplantation and have shown utility in aplastic anemia and paroxysmal hemoglobinuria) And repopulation of the stem cell compartment as normal cells after radiation / chemotherapy in vivo or ex vivo (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy It is also useful in In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the 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, are described in Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486, 1993; including, but not limited to, the assays described in McClanahan et al., Blood 81: 2 903-2915, 1993. Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lymphoper hematopoiesis) are described in Methylcellulose colony forming assays, Fresluley, MG. In Culture of Hematopojetic Cells. RI. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, IK. and Briddell, RA. In Culture of Hematopoietic Cells. RI. Freshney, et al. eds. Vol pp. 23-39, Wiley-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. RI. Freshney, et al. eds. Vol pp-1-21, Wiley-Liss, Inc., New York, NY. 1994: Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T .; In Culture of Hematopoietic Cells RI. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994: Long term culture initiating cell assay, Sutherland, HJ. In Culture of Hematopoietic Cells. RI. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. Includes, but is not limited to, the assays described in 1994. Tissue Proliferation Activity The protein of the present invention has utility in compositions used for the growth or regeneration of bone, cartilage, keys, ligaments and / or nervous tissue, as well as for wound healing and tissue repair, and in the treatment of burns, lacerations and ulcers. Have. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, finds use in healing fractures and cartilage damage or defects in humans and other animals. Such formulations using the protein of the present invention are useful for reducing closed and open fractures and for improving fixation of artificial joints. De novo osteogenesis induced by osteogenic agents contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects and is also useful in cosmetic surgery. The protein of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such agents can provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells, or induce differentiation of osteogenic cell precursors. The protein of the present invention may be used to stimulate osteoporosis or osteoarthritis, for example, by stimulating bone and / or cartilage repair or by blocking the process of tissue destruction mediated by inflammation or inflammatory processes (collagenase activity, osteoclast activity, etc.). May also be useful in the treatment of Another category of tissue development activity that may be due to the proteins of the present invention is key / ligament formation. Proteins of the invention that induce the formation of key / ligament-like or other tissues in an environment in which such tissues are not normally formed can be used to prevent key or ligament laceration, deformation and other key or ligament defects in humans and other animals. Applied to healing, Such formulations using the key / ligament-like tissue inducing protein may be used to prevent damage to the key or ligament tissue as well as to improve the fixation of the key or ligament to bone or other tissue. The de novo key / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of congenital, traumatic, or oncological resection-induced craniofacial defects, and furthermore the attachment of keys or ligaments Or it is also useful in cosmetic surgery for repair. The compositions of the present invention provide an environment for attracting key- or ligament-forming cells, stimulate the growth of key- or ligament-forming cells, and induce the differentiation of key- or ligament-forming cell precursors. Or induce the growth of key / ligament cells or progenitors ex vivo so as to effect tissue repair when returned to vivo. The compositions of the present invention are also useful for keratitis, carpal tunnel syndrome and other key or ligament defects. The compositions of the present invention may include a suitable matrix and / or sequestering agent, such as a carrier well known in the art. The protein of the present invention is useful for the proliferation of nerve cells and the regeneration of nerve and brain tissues, namely, central and peripheral nervous system diseases and neuropathies and mechanical diseases and traumatic diseases (including degeneration, death or trauma to nerve cells or nerve tissues). ) May also be useful. More particularly, diseases of the peripheral nervous system such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, and central nervous system such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager syndrome. The protein may be used to treat systemic diseases. Additional conditions that may be treated according to the present invention include mechanical and traumatic diseases such as spinal cord diseases, cerebrovascular diseases such as head trauma and stroke. Peripheral neuropathy caused by chemotherapy or other medical therapies can be treated with the proteins of the present invention. The protein of the present invention is also useful for promoting more preferable and prompt closure of non-healing wounds including (but not limited to) pressure ulcers, ulcers related to vascular dysfunction, surgical and trauma wounds and the like. It is possible. The protein of the present invention may also be used in other tissues such as organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), and vascular (including vascular endothelium) tissue. Or an activity for promoting the growth of cells constituting such a tissue. Part of the desired effect may be the regeneration of normal tissue by suppressing fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity. The proteins of the present invention may also be useful in protecting or regenerating intestines and treating conditions caused by lung or liver fibrosis, reperfusion injury of various tissues, and systemic cytokine damage. The protein of the present invention may be useful for promoting or suppressing the differentiation of the above-mentioned tissue from precursor tissues or cells; or for suppressing the growth of the above-mentioned tissue. The activity of the protein of the invention may be measured, inter alia, by the following method: Tissue regeneration activity assay is as described in WO 95/16035 (bone, cartilage, key); WO 95/05846 (neural, neuron); Including, but not limited to, the assays described in International Publication WO 91/07491 (skin, endothelium). The assay for wound healing activity was modified by Maibach, HI and Rovee, DT, eds., Year Book Medical Publishers, Inc., Chicago (Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978). ), But not limited thereto. Activin / inhibin activity The protein of the present invention may also exhibit activin- or inhibin-related activity. Inhibins are characterized by their ability to inhibit follicle stimulating hormone (FSH) release, and activins are characterized by their ability to stimulate follicle stimulating hormone (FSH) release. Thus, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces the fertility of female mammals and reduces the spermatogenesis of male mammals. Can be useful as Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the protein of the present invention, as a homodimer or as a heterodimer with subunits of other proteins of the inhibin-β group, can be used to stimulate fertility based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells. May be useful as therapeutic agents that induce See, for example, U.S. Pat. No. 4,798,885. The proteins of the invention may also be useful for improving reproduction in sexually immature mammals, resulting in increased fertility during the life of livestock such as cattle, sheep and pigs. The activity of the protein of the invention may be measured, inter alia, by the following method: Activin / inhibin activity assays are described in Vale et al., Endocrinology 91: 556-572,1972; Ling et al., Nature 321: 779. -782,1986; Vale et al., Nature 321: 77 76-779,1986; Mason et al., Nature 318: 659-663,1985; Forage et al., Proc. Natl. Acad.Sci. USA 83: 3091-3095, 1986, including but not limited to the assays described. Chemotaxis / chemokinetic activity The protein of the present invention can be used as a chemotactic or chemokinetic activity for mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells (eg, acting as a chemokine). ). Chemotactic and chemokinetic proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for treating injuries and other trauma to tissues and for treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent. Certain proteins or peptides have chemotactic activity on a particular cell population and, when stimulable, direct or indirectly direct the movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce adhesion of one cell population to another cell population as well as the ability of the protein to adhere to another cell population. Assays suitable for migration and attachment are described in Current Protocols in Immunology, Ed by JE. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28: Taub et al. J. Clin. Invest. 95: 1370-1376, 1995: Lind et al. APMIS 103: 140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152: 5860-5867, 1994; Immunol. 153: 1762-1768, 1994. Including, but not limited to, hemostatic and thrombolytic activities The proteins of the invention may also exhibit hemostatic or thrombolytic activities. Such proteins are useful in the treatment of various clotting disorders, including genetic disorders such as hemophilia, or promote clotting and other hemostasis in the treatment of injuries caused by trauma, surgery or other causes. The protein of the present invention can dissolve thrombus or inhibit thrombus formation and the symptoms caused by them (eg, myocardial infarction and It may be useful in the treatment and prevention of central nervous system infarction (eg, stroke) The activity of the proteins of the invention may be measured, inter alia, by the following methods: Assays for hemostatic and thrombolytic activities. 26: 31-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991). Schaub, Prostaglandins 35: 467-474, 1988. Including, but not limited to, receptor / ligand activity The protein of the present invention may also be a receptor, receptor ligand or receptor / ligand interaction. Examples of such receptors and ligands are cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cell-cell interactions. Involved in action Receptors and their ligands (including cell adhesion molecules (such as selectins, integrins and their ligands) and receptor / ligand pairs involved in antigen presentation, antigen recognition, development of cellular and humoral immune responses, etc.) But not limited to these. Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors for related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interaction. The activity of the protein of the invention may be measured, inter alia, by the following method: A suitable assay for receptor-ligand activity is described in Current Protocols in Immunology, Ed by JE. Coligan, AM. Kruisbeek, DH. Margulies, EM. Shevach, WS 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 et al., J. Am. Exp. Med. 168: 1145-1156, 1988: Rosenstein et al., J. Am. Exp. Med. 169: 149-160 1989: Stoltenborg et al., J. Am. Immunol. Methods 175: 59-68, 1994: Stitt et al., Cell 80: 661-670, 1995, including but not limited to. Anti-inflammatory activity The protein of the present invention may exhibit anti-inflammatory activity. Anti-inflammatory activity involves stimulating cells involved in the stimulus response, thereby inhibiting or promoting cell-cell interactions (e.g., attachment), thereby enhancing the chemistry of cells involved in the inflammatory process. It is exerted by inhibiting or promoting chemotaxis, inhibiting or promoting cell extravasation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Inflammation symptoms (including chronic or acute symptoms, infection related inflammation (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, Fatal injury by endotoxin, arthritis, hyperacute rejection mediated by complement, nephritis, lung injury induced by cytokines or chemokines, inflammatory bowel disease, Crohn's disease or excessive cytokines such as TNF or IL-1 (Including, but not limited to, diseases arising from production). The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials. Tumor Inhibitory Activity In addition to the activities described above for the immunological treatment or prevention of tumors, the proteins of the present invention may exhibit other antitumor activities. Certain proteins may inhibit tumor growth directly or indirectly (eg, via ADCC). Certain proteins act on tumor tissue or progenitor tissue to inhibit the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) and to inhibit tumor growth. Tumor inhibitory activity may be exhibited by causing the production of a factor, agent or cell type, or by removing or inhibiting a factor, agent or cell type that promotes tumor growth. Other Activities The protein of the invention may exhibit one or more of the following additional activities or effects: killing of infectious agents including but not limited to bacteria, viruses, fungi and other parasites; height Effects on body characteristics including body weight, hair color, eye color, skin or other tissue pigmentation, or the size or morphology of organs or body parts (eg, breast augmentation and vice versa) ); The effects of ingested fat, protein or carbohydrates on digestion; appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders), and violent behavior Effects on inclusive behavioral properties; providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormonal or endocrine activity; Treatment of positive and related diseases; treatment of hyperproliferative diseases (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement); and such proteins acting as antigens in vaccine compositions Or the ability to generate an immune response to other substances or entities that cross-react with such proteins. Administration and Dose The protein of the present invention (which may be from any source, including but not limited to recombinant and non-recombinant methods) is mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition. It may be used in objects. Such compositions may contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend 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, Meg-CSF, Thrombopoietin, stem cells Factors and cytokines such as erythropoietin, lymphokines, or other hematopoietic factors. The pharmaceutical composition may further contain other agents that enhance protein activity or supplement its activity or utility in therapy. Such additional factors and / or agents may be included in a pharmaceutical composition to exert a synergistic effect with the protein of the invention or to minimize side effects. Conversely, specific cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or antithrombotic factors, or by incorporating the protein of the present invention into the formulation of anti-inflammatory agents, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or Side effects of antithrombotic factors or anti-inflammatory agents may be minimized. A protein of the invention may be active in multimers (eg, heterodimers or homodimers) or in complexes with itself or other proteins. Consequently, the pharmaceutical composition of the present invention may contain such a multimeric or complexed form of the protein of the present invention. 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. Protein and / or peptide antigens will deliver a stimulatory signal to B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins. MHC and structurally related proteins, including those encoded by the host cell's class I and class II MHC genes, will serve to present peptide antigens to T lymphocytes. The antigen component is supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, antibodies capable of binding to surface immunoglobulins and other molecules on B cells can be mixed with the pharmaceutical compositions of the present invention. The pharmaceutical composition of the present invention may be in the form of a liposome, in which the protein of the present invention further comprises another pharmaceutically acceptable carrier, an amphipathic agent such as a lipid (in the form of an aggregate such as micelles in an aqueous solution). , Present as an insoluble monolayer, liquid crystal or lamellar layer). Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatide, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposome formulations is within the level of ordinary skill in the art, and is described, for example, in US Pat. It's like it was done. As used herein, the term "therapeutically effective amount" refers to the amount of each active ingredient of a pharmaceutical composition or method that is sufficient to show significant patient benefit, e.g., amelioration of symptoms of such symptoms, healing, increased rate of healing. Means the total amount. When used for an individual active ingredient administered alone, the term refers only to that ingredient. When used in a combination of active ingredients, it refers to the total amount of active ingredients that produces a therapeutic effect regardless of sequential or simultaneous administration. In practicing the therapeutic methods of the invention, a therapeutically effective amount of a protein of the invention is administered to a mammal having the condition to be treated. According to the method of the invention, the protein of the invention may be administered alone or with other therapeutic agents, such as cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the proteins of the invention may be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors. If administered sequentially, the attending physician will determine the appropriate order of administration for combinations of the proteins of the present invention with cytokines, lymphokines, other hematopoietic, thrombolytic or antithrombotic factors. 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 performed by various conventional methods, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. Intravenous injection into a patient is preferred. When a therapeutically effective amount of a protein of the invention is administered orally, the protein of the invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain from about 5 to 95% of a protein of the invention, preferably from about 25 to 90%. When administered in liquid form, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. Liquid form pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of the protein of the present invention, preferably about 1 to 50% of the protein of the present invention. When a therapeutically effective amount of a protein of the present invention is administered by intravenous, intradermal or subcutaneous injection, the protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection include, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride for injection, and Ringer's solution containing lactic acid for injection, Or it should contain other carriers known in the art. The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of the protein of the present invention in the pharmaceutical composition of the present invention depends on the nature and severity of the condition to be treated and the nature of the treatment that the patient has already received. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each patient. First, the attending physician administers a low dose of the protein of the invention and observes the patient's response. Higher doses of the protein of the invention may be administered until the optimal therapeutic effect is obtained, with no further increase in the dose at the time the optimal therapeutic effect is obtained. Various pharmaceutical compositions for carrying out the method of the present invention comprise about 0.01 μg to about 100 mg of the protein of the present invention per kg of body weight (preferably, about 0.1 μg to about 10 mg per kg of body weight, more preferably It should contain from 0.1 μg to about 1 mg of the protein of the invention.The duration of intravenous treatment with the compositions of the invention will vary depending on the severity of the disease to be treated and the condition and response of each patient. It is believed that each application period of the protein of the present invention will be in the range of 12 to 24 hours for continuous intravenous administration, and ultimately the attending physician will administer treatment by intravenous administration using the pharmaceutical composition of the present invention. Animals are immunized with the protein of the present invention to obtain polyclonal and monoclonal antibodies that specifically react with the protein of the present invention. The whole protein or a fragment thereof may be used as an immunogen to obtain such antibodies.The peptide immunogen may further include a cysteine residue at the carboxy terminus, such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, RPMerrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); JLKrstenansky, et.al., FEBS Lett. .211, 10 (1987) Monoclonal antibodies that bind to the protein of the present invention can be useful diagnostic agents for immunodetection of the protein of the present invention Neutralizing antibodies that bind to the protein of the present invention Can be useful as therapeutics for conditions associated with the protein of the present invention, and in the treatment of certain tumors in the treatment of some forms of cancer in which aberrant expression of the protein of the present invention is involved. In the case of cancer cells or white blood cells, neutralizing monoclonal antibodies against the protein of the present invention may be useful in detecting and preventing metastatic spread of cancer cells mediated by the protein of the present invention. With respect to the compositions of the invention useful for the regeneration of the same, the methods of treatment include administering the composition locally, systemically, or locally, such as an implant or device. The composition is, of course, in a pyrogen-free, physiologically acceptable form, and desirably, the composition is encapsulated or injected in a viscous form and delivered to the site of bone, cartilage or tissue damage. Is also good. Topical administration is suitable for wound healing and tissue repair. Therapeutically useful agents other than the protein of the present invention, which may be contained in the above composition, may be alternatively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Preferably, for bone and / or cartilage formation, the composition delivers the protein-containing composition to the site of bone and / or cartilage damage, provides a structure for bone and cartilage development, and more optimally. Will contain a matrix that can be absorbed into the body. Such matrices may be made of materials currently used for other implanted medical devices. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The appropriate formulation will be determined by the particular application of the composition. Possible matrices for the composition may be biodegradable and chemically known calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydride. Other usable materials are biodegradable and biologically well known, such as bone or skin collagen. Further, the matrix may contain pure proteins or extracellular matrix components. Other possible matrices are not biodegradable, such as sintered hydroxyapatite, bioglass, aluminate, or other ceramics, but are chemically known. The matrix may comprise a combination of materials of the above type, for example polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in the composition, for example in calcium-aluminate-phosphate, and may be processed to alter the pore size, particle size, particle shape and biodegradability. A 50:50 (molar weight) cobolimer of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150 to 800 microns is presently preferred. In some applications, it may be useful to use a sequestering agent, such as carboxymethylcellulose or an autologous clot, to prevent dissociation of the protein composition from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose; Most preferred are the cationic salts of (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestering agent useful in the present invention is from 0.5 to 20% by weight, preferably from 1 to 10% by weight, based on the total formulation weight, and this amount prevents desorption of proteins from the polymer matrix. An amount that allows for proper handling of the composition, but not too much so that the progenitor cells infiltrate the matrix, thereby conferring the protein with the opportunity to promote the osteogenic activity of the progenitor cells. Not to be. In a further composition, the protein of the present invention may be mixed with other agents that are beneficial in treating bone and / or cartilage defects, wounds, or tissues. These agents include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). At present, the therapeutic compositions are also valuable for veterinary use. In particular, besides humans, livestock and thoroughbred horses are desirable patients for such treatment with the proteins of the present invention. The rules of administration of the protein-containing pharmaceutical composition used for tissue regeneration may include various factors that alter the action of the protein, such as the tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of the wound, The physician will decide on the type of tissue required (eg, bone), patient age, gender, and diet, severity of infection, duration of administration, and other clinical factors. Dosages may vary depending on the type of matrix used for reconstitution and the inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin-like growth factor I), to the final composition can also affect the dose. Progress can be monitored by periodically assessing tissue / bone growth and / or repair, for example, by X-ray, histomorphological examination and tetracycline labeling. The polynucleotide of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells in vivo or ex vivo and expressed in a mammalian subject. The polynucleotides of the present invention may be administered by other known methods for introducing nucleic acids into cells or organisms, including but not limited to viral vectors or in the form of naked DNA. ). The cells may be cultured and grown ex vivo in the presence of the protein of the present invention, or may have a desired effect on such cells, or may have a desired activity in such cells. The treated cells can then be introduced in vivo for therapeutic purposes. The patents and publications cited herein are considered to be entirely incorporated herein by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/08 C12P 21/02 C C07K 14/47 C12N 15/00 ZNAA C12N 5/10 5/00 B C12P 21/02 A61K 37/02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, H, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Lavery, Edward Earl United States 01876 Massachusetts, Tuxbury, Green Meadow Drive No. 90 (72) Inventor Lacey, Lisa A United States of America 01720 Acton, Mass., School Street 124 (72) Inventor Marberg, David United States 01720 Orton, Mass., Acton, Mass. 72) Inventor Tracy, Maurice United States 02167 Walnut Road 93, Chestnut Hill, Mass. Evans, Cherylinventor 01801 Locust Street Number 41, 111 (72) Invention, Woburn, Mass. 01801 Mass. Spalding, Vicki 01821 Meadowbank Road, Billerica, Mass. 01821

Claims (1)

[Claims]   1. (A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2;   (B) the nucleotide sequence from nucleotide 309 to nucleotide 509 of SEQ ID NO: 2 A polynucleotide comprising a reotide sequence;   (C) Clone BL15 12 deposited under accession number ATCC 98154 A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of   (D) Clone BL1512 deposited under accession number ATCC 98154 Polynucleotide encoding the full-length protein encoded by the cDNA insert of Otide;   (E) Clone BL15 12 deposited under accession number ATCC 98154 A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of   (F) Clone BL1512 deposited under accession number ATCC 98154 Encoding a mature protein encoded by a cDNA insert Otide;   (G) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 Tide;   (H) including a fragment of the amino acid sequence of SEQ ID NO: 3 having biological activity A polynucleotide encoding a protein;   (I) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (f) above Nucleotides;   (J) A polynucleotide encoding a species homolog of the protein of (g) or (h) above. Otide; and   (K) any one of the polynucleotides (a) to (h) under strict conditions Polynucleotide that can hybridize to A composition comprising an isolated polynucleotide selected from the group consisting of:   2. 2. The polynucleotide of claim 1, wherein said polynucleotide is operably linked to an expression control sequence. Composition.   3. A host cell transformed with the composition of claim 2.   4. 4. The host cell of claim 3, wherein said cell is a mammalian cell.   5. (A) growing the culture of the host cell of claim 3 in a suitable medium;   (B) Purifying the protein from the culture And a method for producing a protein.   6. A protein produced by the method of claim 5.   7. 7. The protein of claim 6, comprising a mature protein.   8. A protein comprising an amino acid sequence selected from the group consisting of: A composition comprising a protein substantially free of other mammalian proteins:   (A) the amino acid sequence of SEQ ID NO: 3;   (B) a fragment of the amino acid sequence of SEQ ID NO: 3; and   (C) Clone BL15 12 deposited under accession number ATCC 98154 Amino acid sequence encoded by the cDNA insert of FIG.   9. 9. The composition of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO: 3.   10. 9. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.   11. Administering a therapeutically effective amount of the composition of claim 10 to a mammalian subject. How to prevent, treat or ameliorate a medical condition.   12. Corresponds to the cDNA sequence of SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 4 The gene to do.