JP2001519667A - Secretory expression sequence tags (sESTs) - Google Patents

Abstract

  (57) [Summary] Provided are secreted expressed sequence tags (sESTs) isolated from various human tissues.

Description

DETAILED DESCRIPTION OF THE INVENTION Secreted expression sequence tags (sESTs) Field of the invention The present invention provides novel polynucleotides that are expressed sequence tags (ESTs) for secreted proteins. Background of the Invention Gargantuan's efforts have been used by various research projects to probe random sequence portions of natural cDNAs. The rationale for this approach to gene identification and sequencing is based on two basic principles: (1) that transcribed cDNAs are the products of the most important genes, ie, they are actually expressed in vivo. And (2) efforts to sequence the genes and other parts of the genome of the target organism that are not actually expressed, in areas where therapeutically important genetic information is unlikely to be obtained. It is based on substantially wasting. Thus, high-throughput sequencing focuses only on the part of the genome that is expressed. The randomly obtained cDNA sequences are called "expressed sequence tags" or "ESTs" and can be used to identify long, full length cDNA or genomic sequences from which they are transcribed, and further used as probes for their identification. Can be Although this "shortcut" approach for genomic sequencing saves effort when compared to whole genome sequencing, it also generates many EST sequences that may not be directly useful as proteinaceous therapeutics. To date, most of the discovery of protein-related drugs has focused on the use of secreted proteins to achieve the desired therapeutic effect. In theory, since all expressed proteins are identified by the EST approach, mixtures of secreted proteins (eg, hormones, cytokines and receptors) and non-secreted proteins (eg, metabolic enzymes and cell structural proteins) are used. An EST library containing the ESTs is obtained, but it is not identified which ESTs corresponds to a protein belonging to which category. Consequently, these methods do not adequately satisfy the needs of researchers searching for secreted proteins. Researchers must waste labor and capital to separate the secretory "wheat" from the non-secretory "straw". Commonly owned U.S. Pat. No. 5,536,637, which is hereby incorporated by reference, describes a method for directing genomic sequencing efforts to sequences encoding secreted proteins, and to proteinaceous therapeutic agents. Is of primary interest. U.S. Pat. No. 5,536,637 discloses a "signal sequence trap" for selectively identifying ESTs of secreted proteins (ie, referred to as "secreted expression sequence tags" or "sESTs"). Summary of the Invention The present invention provides sESTs isolated from various human RNA / cDNA sources. In a preferred embodiment, the present invention provides: An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of or a complementary sequence thereof is provided. In another embodiment, the present invention provides: Or an isolated polynucleotide consisting of a nucleotide sequence selected from the group consisting of: In a further embodiment, the present invention provides: An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of or a complementary sequence thereof as an essential component is provided. In yet another embodiment, the invention provides: Or an isolated polynucleotide comprising a nucleotide sequence that hybridizes to a sequence selected from the group consisting of: or a complementary sequence thereof. The present invention also provides a protein encoded by the polynucleotide. Detailed description The nucleotide sequence of the sESTs of the present invention is shown in the sequence listing below. Table 2 shows the "Clone ID Nos" assigned by the applicant to each SEQ ID NO in the sequence listing (SEQ ID NO). Table 2 Each entry pair in this table consists of the sequence number (eg, 1, 2, etc.) followed by the clone ID number for such sequence (eg, B11, B18, etc.). The “clone ID number” of a specific clone is composed of one or two characters followed by a number. The letter indicates the source of efflux from which the sESTs were isolated. Table 3 below shows the various sources from which Applicants' signal sequence traps were obtained. Therefore, the tissue source of the specific sEST sequence can be confirmed in Table 3 by one or two characters used for the corresponding “clone ID number”. For example, the clone named "BA312" was isolated from a library of human (26 year old) placenta, as shown in Table 3 (ie, selectin "BA"). As used herein, the term "polynucleotide" encompasses single- and double-stranded RNAs, DNAs and RNA: DNA hybrids. As used herein, the term “secreted” protein is one that is expressed in a suitable host cell and is transported across a membrane. Transport includes when by a signal sequence in the amino acid sequence. “Secreted” proteins include, but are not limited to, those that are completely secreted from the expressing cells (eg, soluble proteins) and those that are partially secreted (eg, receptors). Also, "secreted" proteins include, but are not limited to, those that are transported through the endoplasmic reticulum membrane. 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 Dowell, et al., J. Am. Amer. Chem. Soc. Cyclization may be carried out 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 the carrier molecule along with the immunoglobulin for many purposes, including increasing the number of valencies at the protein binding site. For example, a protein fragment may be fused via a “linker” to the Fc portion of an immunoglobulin. For bivalent forms of the protein, such fusions can be made to the Fc portion of an IgG molecule. Such fusions may be obtained using other immunoglobulin isotypes. For example, a protein-IgM fusion will yield 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 suitable 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. Using the sequence information disclosed herein, the corresponding gene can be isolated according to known methods. Such methods include preparing probes or primers from the disclosed sequence information to identify and / or amplify genes in a suitable genomic library or other source of genomic material. Where the protein of the invention is membrane-bound (eg, a receptor), the invention also provides soluble forms of such protein. In such a form, some or all of the intracellular and transmembrane domains of the protein are removed so that the protein is sufficiently secreted from the host 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 polynucleotides and proteins are also provided by the invention. As used herein, the term "species homolog" refers to a protein or polynucleotide for a species that is different from the source of the protein or polynucleotide, but that has significant sequence similarity as determined by those skilled in the art. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein, and then screening for an appropriate nucleic acid source from the desired species. The invention also relates to allelic variants of the disclosed polynucleotides, i.e., naturally occurring alternative forms of isolated polynucleotides that encode proteins identical, homologous, or related to the protein encoded by the polynucleotides of the invention. Include. The present invention also encompasses polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein. The invention also encompasses polynucleotides that are capable of hybridizing to the polynucleotides described herein under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions. I do. The following table shows examples of strictness conditions. The very strict conditions are, for example, at least as strict as the conditions A to F, and the strict conditions are, for example, at least as strict as the conditions GL, and the reduced conditions are, for example, For example, it is at least as strict as the conditions M to R. ^‡ : Hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. ^† : SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH in hybridization and wash buffer) _Two PO _Four , And 1.25 mM EDTA, pH 7.4) can be replaced with SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate). After completion of hybridization, washing is performed for 15 minutes. * TB-TR: The hybridization temperature for hybrids expected to be less than 50 base pairs in length is the melting temperature (T _m 5) to 10 ° C. T by the following equation _m Is determined. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (number of A + T bases) +4 (number of G + C bases). For hybrids of 18 to 49 base pairs in length, T _m (℃) = 81.5 + 16.6 (log _Ten [Na ⁺ ]) + 0.41 (% G + C)-(600 / N), where N is the number of bases in the hybrid and the sodium ion concentration in the hybridization buffer ([Na for 1 × SSC ⁺ ] = 0.165M). Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF. Fritsch, and T.W. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, FM. Ausubel et al., Eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are considered to be incorporated herein by reference. Preferably, each such hybridizing polynucleotide has a length of at least 25% (more preferably at least 50%, most preferably at least 75%) of the polynucleotide of the invention to be hybridized, Has at least 60% sequence identity (more preferably, at least 75% identity, most preferably, at least 90% or 95% identity) to the polynucleotide of the invention to be hybridized. Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. The isolated polynucleotide encoding the protein of the present invention can be prepared as described in Kaufman et al., Nucleic Acids Res. The protein may be produced recombinantly by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed in 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. Many suitable expression control sequences are known in the art. General methods for recombinant protein expression are also known and are disclosed in Kaufman, Methods in Enzymology 185, 537-566 (1990) is a typical example. "Operably linked," as defined herein, means that the isolated polynucleotide of the invention and the expression control sequence are placed in a vector or cell and transformed (transfected) with the ligated polynucleotide / expression control sequence. It is meant to be expressed by a host cell. 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 (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primates Includes cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HeLa cells, mouse cells, BHK, HL-60, U937HaK or Jurkat cells. 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 Escherichia coli, NBacillus 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 the baculovirus / insect cell expression system are described, for example, in kit form from In vitrogen, San Diego, California, USA (MaxBac ^R Kits) and such methods are well known in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (which is hereby incorporated by reference). As used herein, insect cells capable of expressing a polynucleotide of the present invention have 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 a culture (ie, medium or cell extract) using known purification processes such as gel filtration and ion exchange chromatography. Protein purification can also be accomplished by using an affinity column containing an agent that will bind to the protein; concanavalin A-agarose, heparin-toyopearl ^R Or Cibacrom blue 3GA Sepharose ^R One or more column steps with an affinity column such as; 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 expression and purification of such fusion proteins are commercially available from Mew Englan 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 in the present invention as "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 have 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 substitutes for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and 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 changes, 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. Applications and biological activities The polynucleotides and proteins of the present invention will 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 applications and usefulness The polynucleotide provided by the present invention can be used for various purposes by the 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 As a probe for subtraction, use a "gene chip" or other To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization, and to generate anti-DNA antibodies, or 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), identifies 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)). The proteins provided by the present invention may be used in assays to determine biological activity, including 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 for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to 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. Nutritional uses The polynucleotides and proteins of the present invention can be used as nutrients 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 solid liquid formulation such as in the form of a powder, pill, solution, suspension or capsule. . 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 A protein of the invention may exhibit cytokine activity, cell growth activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or may induce the production of other cytokines in certain cell populations. 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 can be measured in cell lines (32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G) M + (preeB M +), 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. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays 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: Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., J. Am. Immun ol. 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, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM. and Shevach, EM. In Current Protocols in Immunology. 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 Current Protocols in Imunology. 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. 6.6.1-6.5.5, 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, John 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, Ed by. . 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; 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. Im mun. 11: 405-411, 1981; Takai et al., J. Am. Immunol. 137: 3494-3500, 1986; Takai et al., J. Am. Immunol. 140: 508-512, 1988, but is not limited thereto. Immunostimulatory or suppressive activity The protein of the present 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 particularly, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, such as various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated with 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 needed, 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 for 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 (such as, for example, 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 transplantation, graft rejection 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 may 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 include 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 a stimulatory form 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 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 only a peptide having B7-2-like activity or 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, gene therapy can be used to target tumor cells for transfection in vivo. 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 that lack MHC class I or MHC class II molecules, or that cannot reexpress sufficient amounts of MHC class I or MHC class II molecules, are transformed into MHC class I α chain proteins and β _Two Transfected with a nucleic acid encoding all or part of a microglobulin protein or MHC class II α and β proteins (eg, the cytoplasmic domain of a truncated protein), thereby producing a class I or class II MHC protein Can be expressed on the cell surface. Expression of the 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 the 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 Vitro assays 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: 1968-1974, 1982; Handa et al., J. Am. Immunol. 135: 1564-1572, 1985; Takai et al. 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. 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. Immunol. 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, particularly those that 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 for B cell function as described in In vitro antibody 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. However, it is not limited to these. 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; 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, 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 regulatory 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 that supports colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis. For example, supporting the growth of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing utility in treating various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the growth of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing the resulting myelosuppression; supporting the proliferation of megakaryocytes and then the proliferation of 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 Proliferation of all hematopoietic cells, and therefore of various stem cell diseases (usually those treated by transplantation, which have found utility in aplastic anemia and paroxysmal nocturnal hemoglobinuria) And as normal cells in the stem cell compartment after in vivo or ex vivo radiation / chemotherapy (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy. It is also useful in regrowth. 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; McClanahan et al., Blood 81: 2903-2915, 1993. Not limited to Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lymphoper hematopoiesis) are described in Methylcellulose colony forming assays, Freshney, MG. In Cultu re of Hematopoietic Cells. RI. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. S ci. 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 p p. 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 All en, T.W. 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 proteins of the invention have utility in compositions used for the growth or regeneration of bone, cartilage, keys, ligaments and / or nerve tissue, as well as wound healing and tissue repair, as well as in the treatment of burns, lacerations and ulcers. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, has 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 bone formation 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 may provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells, or induce differentiation of osteogenic cell precursors. The protein of the invention may be osteoporotic 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, ie, 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 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 proteins may be used in the treatment of 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 associated with 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 invention may also be useful in protecting or regenerating the gut and treating conditions resulting from 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). Assays for wound healing activity are described in Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago (modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978)) But not limited thereto. Activin / inhibin activity Also, the protein of the present invention may 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 present 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: 562-572, 1972; Ling et al., Nature 321: 779-. 782, 1986; Vale et al., Nature 321: 776-779, 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. Chemotaxis / chemokinesis activity The protein of the present invention may have chemotactic or chemokinetic activity (eg, acting as a chemokine) on mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells. is there. 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; Johnston et al. J. of Immunol. 153: 1762-1768, 1994, but are not limited thereto. Hemostasis and thrombolytic activity Further, the protein of the present invention may exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various coagulation disorders, including genetic disorders such as hemophilia, or in the treatment of injuries caused by trauma, surgery or other causes and other hemostasis. Can be promoted. The protein of the present invention may be useful for the treatment and prevention of thrombolysis or inhibition of thrombus formation and conditions resulting therefrom (eg, myocardial infarction and central nervous system infarction (eg, stroke)). The activity of the proteins of the invention may be measured, inter alia, by the following method: Assays for haemostatic and thrombolytic activities are described in Linet et al., J. Am. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 467-474, 1988, but not limited thereto. Receptor / ligand activity In addition, the proteins of the present invention may exhibit activity as inhibitors or agonists of receptors, receptor ligands or receptor / ligand interactions. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (cell adhesion Molecules, such as selectins, integrins and their ligands, as well as antigen presentation, antigen recognition, including receptor / ligand pairs involved in the development of cellular and humoral immune responses, and the like. 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: Suitable assays for receptor-ligand activity are described in Current Protocols in Immunology, Ed. E. Coligan, A.S. M. Kruisbeek, D.S. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.2-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6864-6868, 1987; Bierer et al., J. Am. Exp. Med. 168: 1145-1156, 1988; Rosenstein et al. 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, but are not limited thereto. Anti-inflammatory activity The protein of the present invention can 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 immunological treatment or prevention of tumors, the proteins of the 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 tumor precursor 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 proteins 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, weight, Influence (suppression or promotion) on body characteristics including hair color, eye color, skin or other tissue pigmentation, or the size or morphology of organs or body parts (eg, breast augmentation or vice versa); Dietary effects of eaten fat, protein or carbohydrates; behaviors including appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders), and violent behavior (but not limited to them) Effects on 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 related diseases; treatment of hyperproliferative diseases (eg, such as psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement); and such proteins or such proteins acting as antigens in vaccine compositions. Ability to generate an immune response to other substances or entities that cross-react with the protein. 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. May be used. 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 the pharmaceutical composition to exert a synergistic effect with the protein of the present 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. The 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 the 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 stimulatory signals 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 class I and class II MHC genes, will be useful in presenting peptide antigens to T lymphocytes. The antigen component can be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, antibodies that can bind to surface immunoglobulins on B cells and TCRs and other molecules on T 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 contains 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 U.S. Patent Nos. 4,235,871, 4501728, 4837028, and 4,737,323, all of which are hereby incorporated by reference. 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 treatment method of the present invention, a therapeutically effective amount of the protein of the present 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 together 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 present 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 saline solution, dextrose or other saccharide solutions, 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 a protein of the invention, preferably about 1 to 50%. 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 parenterally acceptable protein solutions having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection are, 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 will depend 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, and once the optimal therapeutic effect has been obtained, the dose is not further increased. 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. The duration of each application of the protein of the present invention is considered to be in the range of 12 to 24 hours for continuous intravenous administration. Animals are immunized using 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 and are described, for example, in RP Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); Krstenansky, et.al., FEBS Lett., 211, 10 (1987) Monoclonal antibodies that bind to the protein of the present invention may be useful diagnostic agents for immunodetection of the protein of the present invention. Neutralizing antibodies that bind to the protein of the present invention may be useful as therapeutics for conditions associated with the protein of the present invention, and may also be useful in the treatment of some forms of cancer in which abnormal expression of the protein of the present invention is involved. of In the case of cancer cells or white blood cells, neutralizing monoclonal antibodies against the protein of the invention may be useful in detecting and preventing metastatic spread of cancer cells mediated by the protein of the invention. For compositions of the invention useful for the regeneration of cartilage, keys or ligaments, the methods of treatment include administering the composition locally, systemically, or locally, such as an implant or device. The therapeutic composition used in the present invention is, of course, in a pyrogen-free, physiologically acceptable form, and preferably, the composition is encapsulated or injected as a viscous form into bone, cartilage or tissue. It may be delivered to the damaged site. 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) copolymer 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 methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and alkylcellulose (including hydroxyalkylcellulose), including carboxymethylcellulose, and carboxymethylcellulose (CMC). Most preferred are the cationic salts of 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 detachment of the protein 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 invention may be mixed with other agents that are beneficial for 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, in addition to humans, livestock and thoroughbred horses are the preferred patients for such treatment with the proteins of the present invention. The administration rules of the protein-containing pharmaceutical composition used for the regeneration of the tissue are controlled by various factors that alter the action of the protein, such as the tissue weight desired to be formed, the site of damage, the state 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 have a desired effect on such cells, or 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.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, 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, CH, CN, CU, CZ, DE, DK, E E, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, V N, YU, ZW (72) Inventors Lavery, Edward Earl             United States 01451 Massachusetts             Harvard, 113 Anne Lee Road (72) Inventor Lacey, Lisa Ay             United States 01720 Massachusetts             Acton, School Street 124 (72) Inventor Marberg, David             United States 01720 Massachusetts             Acton, Orchard Drive No. 2 (72) Inventor Tracy, Maurice             United States 02167 Massachusetts             Chestnut Hill, Walcott Low             No. 93 (72) Inventor Spalding, Vicky             United States 01821 Massachusetts             Billalica, Meadowbank Road 11 (72) Inventors Augustino, Michael Jay             United States01810Massachusetts             Andover, Walcott Avenue             26th

Claims (1)

[Claims] 1. The following: An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of or a complementary sequence thereof. 2. The following: An isolated polynucleotide consisting of a nucleotide sequence selected from the group consisting of or a complementary sequence thereof. 3. The following: An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of or a complementary sequence thereof as an essential component. 4. The following: An isolated polynucleotide comprising a nucleotide sequence that hybridizes to a sequence selected from the group consisting of or a complementary sequence thereof. 5. An isolated protein encoded by the isolated polynucleotide of claim 1. 6. An isolated protein encoded by the isolated polynucleotide of claim 2. 7. An isolated protein encoded by the isolated polynucleotide of claim 3. 8. An isolated protein encoded by the isolated polynucleotide of claim 4.