EP0937142A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM

This application is a continuation-in-part of application Ser. No. 08/667,231 , Filed July

9, 1996.

FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.

BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques.

It is to these proteins and the polynucleotides encoding them that the present invention is directed.

SUMMARY OF THE INVENTION In one embodiment, the present 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: l;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 281 to nucleotide 621 ; (c) a polynucleotide composing the nucleotide sequence of the full length protein coding sequence of clone BM46_10 deposited under accession number ATCC 98152,

(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152,

(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BM46_10 deposited under accession number ATCC 98152,

(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152,

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO.2,

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO 2 having biological activity, (l) a polynucleotide which is an allelic variant of a polynucleotide of (a)-

(d) above,

(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above Preferably, such polynucleotide compπses the nucleotide sequence of SEQ ID NO 1 from nucleotide 281 to nucleotide 621 , the nucleotide sequence of the full length protein coding sequence of clone BM46_10 deposited under accession number ATCC 98152, or the nucleotide sequence of the mature protein coding sequence of clone BM46_10 deposited under accession number ATCC 98152 In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152 In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein compπsing the amino acid sequence of SEQ ID NO.2 from ammo acid 1 to ammo acid 79

Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO.1 or SEQ ID NO.3 In other embodiments, the present invention provides a composition compπsing a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of

(a) the ammo acid sequence of SEQ ID NO.2,

(b) the amino acid sequence of SEQ ID NO 2 from amino acid 1 to am o acid 79, (c) fragments of the amino acid sequence of SEQ ID NO:2; and

(d) the amino acid sequence encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 79.

In alternate embodiments, isolate BM46_3 deposited under accession number ATCC 98101 may be substituted for BM46_10 in any of the foregoing.

In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Processes are also provided for producing a protein, which comprise:

(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and (b) purifying the protein from the culture.

The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.

Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.

Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES

Nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application. In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids. The actual nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence.

For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing. Because of the partial ambiguity in reported sequence information, reported protein sequences include "Xaa" designators. These "Xaa" designators indicate either (1 ) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined more accurately). As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplpasmic reticulum.

Clone "BM46 10"

A polynucleotide of the present invention has been identified as clone "BM46_10". BM46_10 was isolated from a human adult muscle cDNA library using methods which are selective for cDNAs encoding secreted proteins. BM46_10 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "BM46_10 protein").

The nucleotide sequence of the 5' portion of BM46_10 as presently determined is reported in SEQ ID NO: 1. What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO:2. The predicted acid sequence of the BM46_10 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Additional nucleotide sequence from the 3' portion of BM46_10, including the polyA tail, is reported in SEQ ID NO:3.

The EcoRI NotI restriction fragment obtainable from the deposit containing clone BM46_ 10 should be approximately 3600 bp.

The nucleotide sequence disclosed herein for BM46_10 was searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. BM46_10 demonstrated at least some identity with ESTs identified as "zb43c09.sl Homo sapiens cDNA clone 306352 3'" (N79027, BlastN) and "H. sapiens EST sequence 008-X" (F19321 , Fasta). Based upon identity, BM46_10 proteins and each identical protein or peptidc may share at least some activity

Deposit of Clone

Clone BM46_10 was deposited on August 23, 1996 with the Amencan Type Culture Collection under accession number 98152

An additional isolate of BM46, BM46_3, was deposited on July 9, 1996 with the Ameπcan Type Culture Collection as part of a composite deposit (with other clones) under accession number ATCC 98101 , from which each clone compπsing a particular polynucleotide is obtainable Each clone has been transfected into separate bacteπal cells (E cob) in this composite deposit Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' cite, EcoRI, 3' cite, NotI) to produce the appropπate fragment for such clone (approximate clone size fragment are identified above) Bacteπal cells containing a particular clone can be obtained from the composite deposit as follows

An o gonucleotide probe or probes should be designed to the sequence that is known for that particular clone This sequence can be deπved from the sequences provided herein, or from a combination of those sequences The sequence of the ohgonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest

Clone Probe Sequence

BM46_10 SEQ ID NO 4

In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/pnmers by a biotmylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotm phosphoramidite ( 1 -dimethoxytπtyloxy- 2-(N-bιotιnyl-4-arrunobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-dιιsopropyl)-phosphoramadιte) (Glen Research, cat no 10-1953))

The design of the ohgonucleotide probe should preferably follow these parameters

(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any,

(b) It should be designed to have a T_m of approx 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C) The ohgonucleotide should preferably be labeled with g-¹²P ATP (specific activity 6000 Ci mmole) and T4 polynucleotide kmase using commonly employed techniques for labeling oligonucleotides Other labeling techniques can also be used Unincorporated label should preferably be removed by gel filtration chromatography or other established methods The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole

The bacteπal culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a steπle culture flask containing 25 ml of steπle L-broth containing ampicillm at 100 μg/ml The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteπological media containing L-broth containing ampicillm at 100 μg/ml and agar at 1 5% in a 150 mm petπ dish when grown overnight at 37°C Other known methods of obtaining distinct, well-separated colonies can also be employed

Standard colony hybπdization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.

The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/hter, 88 2 g Na citrate/liter, adjusted to pH 7 0 with NaOH) containing 0 5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately lO m per 150 mm filter). Preferably, the probe is then added to the hybπdization mix at a concentration greater than or equal to le+6 dpm/mL The filter is then preferably incubated at 65°C with gentle agitation overnight The filter is then preferably washed in 500 mL of 2X SSC/0 5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1 %

SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional The filter is then preferably dπed and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film Other known hybπdization methods can also be employed. The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures The clones can then be veπfied by restπction analysis, hybπdization analysis, or DNA sequencing. Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et i, Bio/Technology K), 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. JJ4, 9245-9253 ( 1 92), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobuiins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunogiobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunogiobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention.

The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form. The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.

Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allehc vaπants of the disclosed polynucleotides or proteins; that is, naturally-occurπng alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al , Nucleic Acids Res _19, 4485-4490 (1991), in order to produce the protein recombinantly Many suitable expression control sequences are known in the art General methods of expressing recombinant proteins are also known and are exemplified m R Kaufman, Methods in Enzymology 185. 537-566 (1990) As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the gated polynucleotide/expression control sequence

A number of types of cells may act 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, other transformed pnmate cell lines, normal dφloid cells, cell strains deπved from in vitro culture of pnmary tissue, pπmary explants, HeLa cells, mouse L cells, BHK, HL- 60, U937, HaK or Jurkat cells Alternatively, it may be possible to produce the protein m lower eukaryotes such as yeast or in prokaryotes such as bacteπa Potentially suitable yeast strains include Saccharomyces cerevtsiae, Schizoiacch rotnyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins Potentially suitable bacteπal strains include Escherichia coh. Bacillus subtihs, Salmonella typhimurium, or any bacteπal strain capable of expressing heterologous proteins If the protein is made in yeast or bactena, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropπate sites, in order to obtain the functional protein Such covalent attachments may be accomplished using known chemical or enzymatic methods

The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system Mateπals and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U S.A. (the MaxBac® kit), and such methods are well known in the art, as descπbed in Summers and Smith, Texas Agπcultural Expenment Station Bulletin No 1555 (1987). incorporated herein by reference As used herein, an insect cell capable of expressing a polynucleotide of the present invention is ' transformed "

The protein of the invention may be prepared by cultuπng transformed host cells under culture conditions suitable to express the recombinant protein The resulting expressed protein may then be punfied from such culture (I e , from culture medium or cell extracts) using known puπfication processes, such as gel filtration and ion exchange chromatography The puπfication of the protein may also include an affinity column containing agents which will bind to the protein, one or more column steps over such affinity resins as concanavahn A- agarose, hepann-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography using such resms as phenyl ether, butyl ether, or propyl ether, or immunoaffinity chromatography

Alternatively, the protein of the invention may also be expressed in a form which will facilitate puπfication For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) Kits for expression and punfication of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively The protein can also be tagged with an epitope and subsequently punfied by using a specific antibody directed to such epitope One such epitope ("Flag") is commercially available from Kodak (New Haven, CT) Finally, one or more reverse-phase high performance liquid chromatography (RP-

HPLC) steps employing hydrophobic RP-HPLC media, e g , silica gel having pendant methyl or other aliphatic groups, can be employed to further puπfy the protein Some or all of the foregoing puπfication steps, in vanous combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein The protein thus punfied is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein "

The protein of the invention may also be expressed as a product of transgenic animals, e g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are characteπzed by somatic or germ cells containing a nucleotide sequence encoding the protein The protein may also be produced by known conventional chemical synthesis

Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art The synthetically-constructed protein sequences, by virtue of shaπng pπmary, secondary or tertiary structural and or conformational characteπstics with proteins may possess biological properties in common therewith, including protein activity Thus, they may be employed as biologically active or immunological substitutes for natural, punfied proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.

4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

USES AND BIOLOGICAL ACTIVITY

The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

Research Uses and Utilities

The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.

Nutritional Uses

Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

Cvtokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/1 1, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI , 123, TI 165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Assays forT-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341 , 1991 ; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756- 1761 , 1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. 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, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1 -6.3.12, John Wiley and Sons. Toronto. 1991 ; deVrie et al., J. Exp. Med. 173: 1205-121 1, 1991 ; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931 -2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1 -6.6.5, John Wiley and Sons, Toronto. 1991 ; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 1 1 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. 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, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1 , John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-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. Immun. 11 :405-411, 1981 ; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders

(including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis. myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention. Using the proteins of the invention it may also be possible to immune responses, 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 preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen- specific and persists after exposure to the toierizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the toierizing agent.

Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1 , B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans Examples of appropπate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as descπbed in Lenschow et al . Science 257 789-792 (1992) and Turka et al , Proc Natl Acad Sci USA, 89 1 1 102- 11 105 (1992) In addition, muπne models of GVHD (see Paul ed , Fundamental Immunology, Raven Press, New York, 1989, pp 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases Many autoimmune disorders are the result of mappropπate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoanti bodies involved in the pathology of the diseases Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms Administration of reagents which block costimulation of T cells by disrupting receptor ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-deπved cytokines which may be involved in the disease process Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well- charactenzed animal models of human autoimmune diseases Examples include muπne expeπmental autoimmune encephalitis, systemic lupus erythmatosis in MRUlpr/lpr mice or NZB hybnd mice, munne autoimmune collagen arthπtis, diabetes melhtus in NOD mice and BB rats, and muπne expeπmental myasthenia gravis (see Paul ed , Fundamental Immunology, Raven Press, New York, 1989, pp 840-856) Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy Upregulation of immune responses may be the form of enhancing an existing immune response or eliciting an initial immune response For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as descπbed herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient The infected cells would now be capable of de veπng a costimulatory signal to, and thereby activate, T cells in vivo

In another application, up regulation or enhancement of antigen function (preferably

B lymphocyte antigen function) may be useful in the induction of tumor immunity Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject If desired, the tumor cell can be transfected to express a combination of peptides For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-hke activity alone, or in conjunction with a peptide having B7-l -lιke activity and/or B7-3-hke activity The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo

The presence of the peptide of the present invention having the activity of a B lymphocyte antιgen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e g , a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and β₂ microglobu n protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropπate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g , B7-1 , B7-2, B7- 3) induces a T cell mediated immune response against the transfected tumor cell Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invaπant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject The activity of a protein of the invention may, among other means, be measured by the following methods.

Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those descπbed in Current Protocols in Immunology, Ed by J. E Coligan, A.M Kruisbeek, D.H Marguhes, E.M. 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. Immunol. 128:1968-1974, 1982; Handa et al., J Immunol 135: 1564-1572, 1985; Takai et al., J. Immunol. 137.3494-3500, 1986; Takai et al., J Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci USA 78.2488-2492, 1981 ; Herrmann et al., J Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol 135 1564-1572, 1985, Takai et al., J. Immunol. 137 3494-3500, 1986; Bowmanet al., J Virology 61 : 1992-1998; Takai et al., J. Immunol. 140:508-512, 1988, Bertagnolli et al., Cellular Immunology 133:327-341, 1991 ; Brown et al., J Immunol. 153:3079-3092, 1994 Assays for T-cell -dependent immunogiobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Marguhes, E.M. 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), Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,

J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendπtic cell-dependent assays (which will identify, among others, proteins expressed by dendπtic cells that activate naive T-cells) include, without limitation, those descπbed in-

Guery et al., J. Immunol 134:536-544, 1995, Inaba et al., Journal of Expeπmental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al , Journal of Expeπmental Medicine 182 255-260, 1995, Nair et al , Journal of Virology 67 4062-4069, 1993, Huang et al , Science 264 961 -965, 1994, Macatonia et al , Journal of fcxpeπmental Medicine 169 1255-1264, 1989, Bhardwaj et al , Journal of Clinical Investigation 94 797-807, 1994, and Inaba et al , Journal of Expenmental Medicine 172 631 640, 1990

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those descπbed in 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 Immunology 145 4037-4045, 1990, Zamai et al , Cytometry 14 891 -897, 1993, Gorczyca et al , International Journal of Oncology 1 639-648, 1992

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those descπbed in. Antica et al , Blood 84 1 1 1 -1 17, 1994, Fine et al., Cellular Immunology 155 11 1-122, 1994, Galy et al , Blood 85 2770-2778, 1995, Toki et al , Proc Nat Acad Sci USA 88 7548-7551 , 1991

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e g in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating vanous anemias or for use in conjunction with lrradiation chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells, in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (l e , traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression, supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of vaπous platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of matuπng to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in vaπous stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglob uπa), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods:

Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those 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.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-591 1 , 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. 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. R.I. 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. R.I. Freshney, et al eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints De novo bone formation induced by an osteogenic agent contπbutes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery

A protein of this invention may also be used in the treatment of peπodontal disease, and in other tooth repair processes Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthntis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc ) mediated by inflammatory processes Another category of tissue regeneration activity that may be attπbutable to the protein of the present invention is tendon/ligament formation A protein of the present invention, which induces tendon/ligament-hke tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals Such a preparation employing a tendon/ligament-hke tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repaiπng defects to tendon or ligament tissue De novo tendon/ligament-hke tissue formation induced by a composition of the present invention contπbutes to the repair of congenital, trauma induced, or other tendon or ligament defects of other ongm, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair The compositions of the invention may also be useful m the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or faster closure of non- healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.

A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. W091/07491 (skin, endothelium ).

Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71 -1 12 (Maibach, HI and Rovee, DT, eds ), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71 :382-84 (1978).

Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin inhibin activity include, without limitation, those 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.

Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed oπentation or movement of such cell population Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis The activity of a protein of the invention may, among other means, be measured by the following methods

Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population Suitable assays for movement and adhesion include, without limitation, those descπbed in Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, E M Shevach, W Strober, Pub Greene Publishing Associates and Wiley-Interscience (Chapter 6 12, Measurement of alpha and beta Chemok es 6 12 1-6 12 28, Taub et al J Chn 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

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolytic activity As a result, such a protein is expected to be useful in treatment of vaπous coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e g , stroke)

The activity of a protein of the invention may, among other means, be measured by the following methods

Assay for hemostatic and thrombolytic activity include, without limitation, those descπbed in Lmet et al , J Chn Pharmacol 26 131-140, 1986, Burdick et al , Thrombosis Res 45 413-419, 1987, Humphrey et al , Fibπnolysis 5 71 -79 (1991 ), Schaub, Prostaglandins 35 467-474, 1988

Receptor/Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integπns and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses) Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions

The activity of a protein of the invention may, among other means, be measured by the following methods

Suitable assays for receptor-ligand activity include without limitation those descnbed in Current Protocols in Immunology, Ed by J E Coligan, A M Kruisbeek, D H Marguhes, E M Shevach, W Strober, 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, Bierer et al , J Exp Med 168 1 145-1 156, 1988, Rosenstein et al , J Exp Med 169 149-160 1989, Stoltenborg et al , J Immunol Methods 175 59-68, 1994, Stitt et al , Cell 80 661 -670, 1995

Anti -Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatory activity The anti- mflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), lschemia- repertusion injury, endotoxin lethality, arthπtis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1 Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or mateπal

Tumor Inhibition Activity

In addition to the activities descπbed above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities A protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC) A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth

Other Activities

A protein of the invention may also exhibit one or more of the following additional activities or effects inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteπa, viruses, fungi and other parasites, effecting (suppressing or enhancing) bodily characteπstics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape), effecting biorhythms or cancadic cycles or rhythms, effecting the fertility of male or female subjects, effecting the metabolism, catabo sm, anabolism, processing, utilization, storage or elimination of dietary fat, hpid, protein, carbohydrate, vitamins, minerals, cofactors or other nutntional factors or component(s); effecting behavioral charactenstics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors, providing analgesic effects or other pain reducing effects, promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages, hormonal or endocπne activity, in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases, treatment of hyperprohferative disorders (such as, for example, psonasis), lmmunoglobulin-hke activity (such as, for example, the ability to bind antigens or complement), and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another matenal or entity which is cross-reactive with such protein

ADMINISTRATION AND DOSING

A protein of the present invention (from whatever source deπved, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable earner Such a composition may also contain (in addition to protein and a earner) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other mateπals well known in the art The term "pharmaceutically acceptable" means a non-toxic matenal that does not interfere with the effectiveness of the biological activity of the active mgredιent(s) The charactenstics of the earner will depend on the route of administration The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, EL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1 , TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent A protein of the present invention may be active in multimers (e g , heterodimers or homodimers) or complexes with itself or other proteins As a result, pharmaceutical compositions of the invention may compnse a protein of the invention in such multimeπc or complexed form

The pharmaceutical composition of the invention may be in the form of a complex of the protem(s) of present invention along with protein or peptide antigens The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes B lymphocytes will respond to antigen through their surface immunogiobulin receptor T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antιgen(s) to T lymphocytes The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention

The pharmaceutical composition of the invention may be in the form of a hposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable earners, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution Suitable lipids for hposomal formulation include, without limitation, monoglyceπdes, diglyceπdes, sulfatides, lysolecithin, phosphohpids, saponin, bile acids, and the like Preparation of such hposomal formulations is within the level of skill in the art, as disclosed, for example, in U S Patent No 4,235,871 , U S Patent No 4,501,728, U S Patent No 4,837,028, and U S Patent No 4,737,323, all of which are incorporated herein by reference As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, l e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, senally or simultaneously

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors When co- administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokιne(s), lymphokιne(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially If administered sequentially, the attending physician will decide on the appropπate sequence of admimstenng protein of the present invention in combination with cytokιne(s), lymphokιne(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present 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 additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition 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 from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.

When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, 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 due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further It is contemplated that the vanous pharmaceutical compositions used to practice the method of the present invention should contain about 0 01 μg to about 100 mg (preferably about 0 lμg to about 10 mg, more preferably about 0 1 μg to about 1 mg) of protein of the present invention per kg body weight The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration Ultimately the attending physician will decide on the appropπate duration of intravenous therapy using the pharmaceutical composition of the present invention

Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH) Methods for synthesizing such peptides are known in the art, for example, as in R P Memfield, J Amer Chem Soc 8_5, 2149-2154 ( 1963), J L Krstenansky, et al, FEBS Lett 21_1, 10 (1987) Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the lmmunodetection of the protein Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein

For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administenng the composition topically, systematically, or locally as an implant or device When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage Topical administration may be suitable for wound healing and tissue repair Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as descnbed above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention Preferably for bone and or cartilage formation, the composition would include a matπx capable of deliveπng the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matnces may be formed of materials presently in use for othei implanted medical applications

The choice of matπx matenal is based on biocompatibihty, biodegradabi ty, mechanical properties, cosmetic appearance and interface properties The particular application of the compositions will define the appropπate formulation Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tπcalciumphosphate, hydroxyapatite, polylactic acid, polyglyco c acid and polyanhydndes Other potential mateπals are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matnces are compπsed of pure proteins or extracellular matπx components Other potential matnces are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, alummates, or other ceramics Matnces may be compπsed of combinations of any of the above mentioned types of matenal, such as polylactic acid and hydroxyapatite or collagen and tπcalciumphosphate The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabihty Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycohc acid in the form of porous particles having diameters ranging from 150 to 800 microns In some applications, it will be useful to utilize a sequesteπng agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matπx. A preferred family of sequestenng agents is cellulosic mateπals such as alky lcellu loses

(including hydroxyalkylcellu loses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequesteπng agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol)

The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1 -10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matπx and to provide appropπate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matnx, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.

These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin- like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.

The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with 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, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling. Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

Patent and literature references cited herein are incorporated by reference as if fully set forth. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Jacobs, Kenneth McCoy, John LaVallie, Edward Racie, Lisa Merberg, David Treacy, Maurice Spaulding, Vikki

(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Genetics Institute, Inc.

(B) STREET: 87 CambridgePark Drive

(C) CITY: Cambridge

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02140

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Brown, Scott A.

(B) REGISTRATION NUMBER: 32,724

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 498-8224

(B) TELEFAX: (617) 876-5851

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 621 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

ATAACATCTC CCCAGACCCA GAAAAACAGA AAGCTCCACA GAAATTAAAT GTTGAAGAGA 60

AACTCTCAAA GGAAGTTACA GAAGAAAACT ATCTCTTTCC CAGTAAGTTC AGTGGAAAGT 120

GCACTAGAAC ATGAATATGA CTTGGTGAAT TAGATGAAAG TTTTTATGGA CCAGAAAAGG 180

CCACAACATA TTATCTCATC CAGAGACCCA AAGCCAAAAC TCAGCTGACA GGAATGTTTC 240

AAAGGACACA AAGAGAGATG TGGACTCAAA GTCACCGGGG ATGCCTTTAT TTGAAGCAGA 300

GGAAGGAGTT CTATCACGAA CCCAGATATT TCCTACCACT ATTAAAGTCA TTGATCCAGA 360

ATTTCTGGAG GAGCCACCTG CACTTGCATT TTTATATAAG GATCTGTATG AAGAAGCAGT 420

TGGAGAGAAA AAGAAGGAAG AGGAGACAGC TTCTGAAGGT GACAGTGTGA ATTCTGAGGC 480

ATCATTTCCC AGCAGAAATT CTGACACTGA TGATGGAACA GGAATATATT TTGAGAAGTN 540

CATACTCAAA GATGACATTC TCCATGACAC ATCTCTAACT CAAAAGGACC ANGGCCAAGG 600

TCTGGAAAAA AAACAANTTG G 621 (2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 112 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Pro Leu Phe Glu Ala Glu Glu Gly Val Leu Ser Arg Thr Gin lie 1 5 10 15

Phe Pro Thr Thr lie Lys Val lie Asp Pro Glu Phe Leu Glu Glu Pro 20 25 30

Pro Ala Leu Ala Phe Leu Tyr Lys Asp Leu Tyr Glu Glu Ala Val Gly 35 40 45

Glu Lys Lys Lys Glu Glu Glu Thr Ala Ser Glu Gly Asp Ser Val Asn 50 55 60

Ser Glu Ala Ser Phe Pro Ser Arg Asn Ser Asp Thr Asp Asp Gly Thr 65 70 75 80 Gly lie Tyr Phe Glu Lys Xaa lie Leu Lys Asp Asp lie Leu His Asp 85 90 95

Thr Ser Leu Thr Gin Lys Asp Xaa Gly Gin Gly Leu Glu Lys Lys Gin 100 105 110

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 315 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

TCTGTTTTAC TGGTAAAGGA AATCCTCTGA TGGACAGGTC AGAGTGAAGG AAGGTTGTGC 60

TGGTAAGACA TNTCTGANGA AGAGCCATGG ATGCTTTCCA CAAAATGTCA CCTCGCTGCA 120

CTAAAGGATG ATGAATCCTA ATCATTAAAG GAATTGTTTC AGCTGATTTA AATTTATAAT 180

GAACTCTTTT GTAATAATGT ATACTGTAGA ACATGAGTCT CTCCTCCCTA AAATTTTAAA 240

TGTAGAAAAG TGCTATATAT TAGAAATTTC CATTTTGTTA AATAAATGGT TAGAGTCTAT 300

AAAAAAAAAA AAAAA 315 (2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : GNCTCCTCCA GAAATTCTGG ATCAATGAC 29

Claims

What is claimed is:

1. A composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 ;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 281 to nucleotide 621 ;

(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone BM46_ 10 deposited under accession number ATCC 98152;

(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152;

(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BM46_10 deposited under accession number ATCC 98152;

(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;

(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; and

(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above .

2. A composition of claim 1 wherein said polynucleotide is operably linked to an expression control sequence.

3. A host cell transformed with a composition of claim 2.

4. The host cell of claim 3, wherein said cell is a mammalian cell.

5. A process for producing a protein, which comprises:

(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and

(b) purifying the protein from the culture 6 A protein produced according to the process of claim 5

7 The protein of claim 6 compπsing a mature protein

8 A composition compπsing a protein, wherein said protein compπses an amino acid sequence selected from the group consisting of

(a) the amino acid sequence of SEQ ID NO 2,

(b) the amino acid sequence of SEQ ID NO 2 from ammo acid 1 to amino acid 79,

(c) fragments of the amino acid sequence of SEQ ID NO 2, and

(d) the amino acid sequence encoded by the cDNA insert of clone BM46_10 deposited under accession number ATCC 98152, the protein being substantially free from other mammalian proteins

9 The composition of claim 8, wherein said protein compπses the amino acid sequence of SEQ ID NO 2

10 The composition of claim 8, further comprising a pharmaceutically acceptable earner

1 1 A method for preventing, treating or ameliorating a medical condition which comprises administenng to a mammalian subject a therapeutically effective amount of a composition of claim 10

12 The gene corresponding to the cDNA sequence of SEQ ID NO 1 or SEQ ID NO 3