This application is a continuation-in-part of application Ser. No. 08/721 ,926, filed

September 27, 1996, which was a continuation-in-part of application Ser. No. 08/664,596, filed June 17, 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) 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 14 to nucleotide 433;

(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone C195_l deposited under accession number ATCC 98079;

(d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone C195_l deposited under accession number ATCC 98079;

(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone C195_l deposited under accession number ATCC 98079;

(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone C 195_ 1 deposited under accession number ATCC 98079;

(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 comprising the nucleotide sequence of the full length protein coding sequence of isolate C195_4 deposited under accession number ATCC 98192;

(j) a polynucleotide encoding the full length protein encoded by the cDNA insert of isolate C195_4 deposited under accession number ATCC 98192; (k) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of isolate C195_4 deposited under accession number ATCC 98192;

(1) a polynucleotide encoding the mature protein encoded by the cDNA insert of isolate C195_4 deposited under accession number ATCC 98192; (m) a polynucleotide comprising the nucleotide sequence of SEQ ID

NO:6;

(n) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 14 to nucleotide 733;

(o) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:7;

(p) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:7 having biological activity;

(q) a polynucleotide which is an allelic variant of a polynucleotide of (a)- (e) or (i)-(n) above;

(r) a polynucleotide which encodes a species homologue of the protein of (g), (h), (o) or (p) above .

Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 from nucleotide 14 to nucleotide 433; the nucleotide sequence of the full length protein coding sequence of clone C195_l deposited under accession number ATCC 98079; or the nucleotide sequence of the mature protein coding sequence of clone C195_l deposited under accession number ATCC 98079. In other preferred embodiments, the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone C195_l deposited under accession number ATCC 98079. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 52 to amino acid 140.

Other preferred embodiments comprise those in which the polynucleotide comprising the nucleotide sequence of SEQ ID NO:6; the nucleotide sequence of SEQ ID NO:6 from nucleotide 14 to nucleotide 733; a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:7; or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:7 having biological activity;

Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO:3 and the gene corresponding to the cDNA sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises 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 amino acid 52 to amino acid 140;

(c) fragments of the amino acid sequence of SEQ ID NO:2;

(d) the amino acid sequence encoded by the cDNA insert of clone C195_l deposited under accession number ATCC 98079;

(e) the amino acid sequence of SEQ ID NO:7; (f) the amino acid sequence of SEQ ID NO:7 from amino acid 1 to amino acid 140;

(g) fragments of the amino acid sequence of SEQ ID NO:7; and (h) the amino acid sequence encoded by the cDNA insert of isolate C195_4 deposited under accession number ATCC 98192;

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 52 to amino acid 140. In other preferred embodiments, such protein comprises the amino acid sequence of SEQ ID NO:7 or the amino acid sequence of SEQ ID NO:7 from amino acid 1 to amino acid 140.

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.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation of the pED6 and pNotS vectors used for deposit of clones disclosed herein.

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 "Cl 95 1 "

A polynucleotide of the present invention has been identified as clone "C195_l " C195_l was isolated from a human PBMC cDNA library using methods which are selective for cDNAs encoding secreted proteins C195_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "C195_l protein")

The nucleotide sequence of the 5' portion of C195_l 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 amino acid sequence of the

C195_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 2 Additional nucleotide sequence from the 3' portion of C 195_1 , including the polyA tail, is reported in SEQ ID NO:3

The EcoRI/NotI restriction fragment obtainable from the deposit containing clone C195_l should be approximately 1350 bp

The nucleotide sequence disclosed herein for C195_l was searched against the

GenBank database using BLASTA/BLASTX and FASTA search protocols C195J demonstrated at least some identity with two ESTs identified as "ygl lg06 rl Homo sapiens cDNA clone 31937 5'" (BiastN, accession number R1721 1) and "yml5fl2 rl Homo sapiens cDNA clone 48025 5'" (BiastN, accession number HI 1976) C195J also contains a WD

motif Based upon identity, C195_l proteins and each identical protein or peptide may share at least some activity

Clone "C195_4" is an additional isolate of C195_l The nucleotide sequence of the 5' portion of C195_4 as presently determined is reported in SEQ ID NO 5 Additional internal nucleotide sequence for C195_4 is reported in SEQID NO 6 What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO 7 The predicted ammo acid sequence of the C195_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO 7 Additional nucleotide sequence from the 3' portion of C195_4, including the polyA tail, is reported in SEQ ID NO 8 Search of the database using the additional sequence from C 195_4 revealed at least some identity with an mRNA for human FAN protein (X96586, Fasta), with which C195_l or C195_4 protein may share some activity

Deposit of Clones

Clone C195_l was deposited on June 15, 1996 with the American Type Culture Collection under accession number ATCC 98079, from which the clone is obtainable This is a composite deposit compnsing a mixture of cells containing C195_l with cell containing other clones. Each clone has been transfected into separate bacteπal cells (E colt) in this composite deposit An additional isolate of C195_l (identified as "C195_4") was deposited on September 26, 1996 with the Ameπcan Type Culture Collection under accession number ATCC 98192, from which the clone is also obtainable

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 appropnately sized fragment for such clone (approximate clone size fragment are identified below) Each clone was deposited in either the pED6 or pNotS vector depicted in Fig 1 In some instances, the deposited clone can become "flipped" (I e , in the reverse onentation) in the deposited isolate In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI However, NotI will then produce the 5' cite and EcoRI will produce the 3' cite for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited

Bacteπal cells containing a particular clone can be obtained from the composite deposit as follows

An oligonucleotide 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 oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.

5 Clone Probe Sequence

C195_l SEQ ID NO:4

C195_4 SEQ ID NO:4

In the sequences listed above which include an N at position 2, that position is occupied in 10 preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy- 2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-d iisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).

The design of the oligonucleotide probe should preferably follow these parameters: 15 (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 oligonucleotide should preferably be labeled with g- ³² P ATP (specific activity 6000 20 Ci/mmole) and T4 polynucleotide kinase 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

25 approximately 4e+6 dpm/pmole.

The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin 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.

30 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 bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.

Standard colony hybridization 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 Nd citrate/liter, adjusted to pH 7 0 with NaOH) containing 0 5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybndization 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 59c 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 hybndization 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 verified by restπction analysis, hybndization 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 cychzed using known methods, for example, as descπbed in H.U. Saragovi, et al., Bio/Technology J_0, 773-778 (1992) and in R S. McDowell, et al., J. Amer. Chem. Soc. 1 14. 9245-9253 (1992), both of which are incorporated herein by reference Such fragments may be fused to earner molecules such as immunoglobulins 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 immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin 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 m 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 pnmers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libranes 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 pnmers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.

The invention also encompasses allelic 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. J _. 9, 4485-4490 (1991 ), in order to produce the protein recombmantly Many suitable expression control sequences are known in the art General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymoiogy 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated withm a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated 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 diploid cells, cell strains deπved from in vitro culture of pπmary 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 in lower eukaryotes such as yeast or in prokaryotes such as bacteria Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Khtyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins Potentially suitable bacterial 5 strains include Eschenchia coli, Bacillus subtihs, Salmonella typhimurtum, or any bacteπal strain capable of expressing heterologous proteins If the protein is made in yeast or bacteπa, 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

10 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

15 descπbed in Summers and Smith, Texas Agncultural 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 cultunng transformed host cells under culture conditions suitable to express the recombinant protein The resulting expressed protein

20 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 punfication 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 concanavalin A- agarose, hepann-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving

25 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 punfication 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

30 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 perfonnance 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 punfy the protein Some or all of the foregoing punfication steps, in vaπous 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 shanng pπmary, secondary or tertiary structural and/or conformational charactenstics 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 characteπzed by amino acid sequences similar to those of punfied 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 ammo 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 denvatives of the sequences of proteins which would be expected to retain protein activity m 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 descπbed for proteins of the present invention may be 5 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

10 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, characteπzation 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

15 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 hybndize and thus discover novel, related DNA sequences; as a source of information to denve PCR pnmers for genetic fingerpπnting; as a probe to "subtract-out" known sequences in the process of discovenng other novel

20 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-hgand interaction), the

25 polynucleotide can also be used m interaction trap assays (such as, for example, that descπbed 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

30 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 35 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. 5 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

10 and T. Maniatis eds., 1989, and "Methods in Enzymoiogy: 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 15 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 20 microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cell proliferation (either

25 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

30 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, Tl 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 for T-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 5 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

10 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.

15 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 ; deVries et al., J. Exp. Med. 173:1205-121 1 , 1991 ; Moreau et al., Nature 336:690-692,

20 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. 25 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,

30 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 35 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 1 1 405-41 1 , 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 descπbed herein A protein may be useful in the treatment of vaπous 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 bactenal or fungal infections, or may result from autoimmune disorders More specifically, infectious diseases causes by viral, bactenal, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteπa, Leishmania spp , malaπa spp and vaπous 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 arthπtis, 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 m that it is generally antigen specific and persists after exposure to the toleπzmg 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 tolerizing 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 5 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

10 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

15 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

20 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 appropriate systems which can be used include allogeneic cardiac grafts in rats

25 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 described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 59: 1 1 102- 1 1105 (1992). In addition, murine 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

30 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 inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of

35 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-derived 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- characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental 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 in 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 described 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 delivering 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-like activity alone, or in conjunction with a peptide having B7-l -like

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 antigen(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 α chain protein and β ₂ microglobulm protein or an MHC class II α chain protein and an MHC class π 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 π 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 desenbed 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); 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 a!., Cellular Immunology 133.327-341 , 1991 ; Brown et al., J. Immunol. 153:3079-3092, 1994

Assays for T-cell-dependent immunoglobulin 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 Mahszcwski, 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 descπbed 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, 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 descnbed 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 Expenmental 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 Expeπmental Medicine 169:1255-1264, 1989, Bhardwaj et al , Journal of Clinical Investigation 94 797-807, 1994; and Inaba et al., Journal of Expeπmental Medicine 172 631 - 640, 1990

Assays for lymphocyte survival7apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described 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 described in: Antica et al , Blood 84- 11 1-117, 1994, Fine et al , Cellular Immunology 155:1 1 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 vaπous anemias or for use in conjunction with irradiation/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 (1 e , traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression, in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of vanous 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 hemoglobinuπa), as well as in repopulatmg the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (1 e , in conjunction with bone marrow transplantation or with peπpheral 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 descπbed 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 descπbed 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, Pπmitive hematopoietic colony forming cells with high prohferative potential, McNiece, I K and Bπddell, 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 contributes 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 periodontal 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 osteoarthritis, 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 attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like 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-like 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 repairing defects to tendon or ligament

tissue De novo tendon/ligament-like tissue formation induced by a composition of the present invention contnbutes 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 5 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 in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects The compositions

10 may also include an appropπate matnx and/or sequestenng agent as a earner 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 penpheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders,

15 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 penpheral nervous system, such as penpheral nerve injuπes, penpheral 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

20 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 Penpheral 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-

25 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

30 (including vascular endothelium) tissue, or for promoting the growth of cells compnsing such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarπng 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 vaπous tissues, and conditions resulting from systemic cytokine damage

A protein of the present invention may also be useful for promoting or inhibiting 5 differentiation of tissues descπbed above from precursor tissues or cells, or for inhibiting the growth of tissues descπbed 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 descπbed in 10 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 descπbed in Winter, Epidermal Wound Healing, pps 71 -1 12 (Maibach, HI and Rovee, DT, eds ), Year 15 Book Medical Publishers, Inc , Chicago, as modified by Eaglstein and Mertz, J Invest Dermatol 71 :382-84 (1978)

Activin/Inhibm Activity

A protein of the present invention may also exhibit activm- or inhibin-related 20 activities Inhibins are characteπzed by their ability to inhibit the release of follicle stimulating hormone (FSH), while activms and are charactenzed 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 inhibm α family, may be useful as a contraceptive based on the ability of mhibms to decrease fertility in female mammals and decrease spermatogenesis

25 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 activm molecules in stimulating FSH release from cells of the anterior pituitary See, for example, United States Patent 4,798,885 A

3 0 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. 5

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

10 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.

15 A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation 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

20 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 25 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 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 6.12, Measurement of alpha and beta Chemokines 30 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.

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 various 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 5 of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of condiuons 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 10 Assay for hemostatic and thrombolytic activity include, without limitation, those descnbed in Linet 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, Prostaglandms 35 467-474, 1988

15 Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/hgand 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

20 interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, mtegnns and their ligands) and receptor/hgand 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/hgand interaction A protein of the present invention

25 (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/hgand interactions

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

Suitable assays for receptor-hgand activity include without limitation those descπbed

30 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 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 35 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 inflammatory 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- reperfusion injury, endotoxin lethality, arthπtis, complement-mediated hyperacute re _j ection, nephπtis, 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 matenal

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 rumor 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 caπcadic cycles or rhythms, effecting the fertility of male or female

sub _j ects, effecting the metabolism, catabohsm, anabohsm, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutπtional 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 endocrine activity, in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases, treatment of hyperproliferative disorders (such as, for example, psoriasis), immunoglobulin-like 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 carrier 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, JE-6, JE-7, IL-8, IL-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 oi homodimers) or complexes with itself or other proteins As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeπc or complexed form 5 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 immunoglobulin receptor T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by

10 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 punfied 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

15 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

20 micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution Suitable lipids for hposomal formulation include, without limitation, monoglyceπdes, diglycendes, sulfatides, lysolecithin, phospholipids, 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

25 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, i.e , treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of

30 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, seπally or simultaneously

In practicing the method of treatment or use of the present invention, a therapeutically 35 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 m combination with other therapies such as treatments employing cytokines, lymphokmes or other hematopoietic factors When co- administered with one or more cytokines, lymphokmes 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 appropriate sequence of administering 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 vanety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intrapeπtoneal, 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 m 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 Chloπde 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 various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg 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 appropriate 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. Merrifield, J. Amer.Chem.Soc. 85 _> 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 2JJ_, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection 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 administering 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 described 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 matrix capable of delivering 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 matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.

Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic 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 sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering 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 matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, 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 detemiined 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.

5 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: 8

(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: 433 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

GTCGACCTTG AAGATGTTTT CTAAAGAATC AAAAATGCTA CAAAGAAGTA TATCATTTTC 60

AAATATGGCT TTATCGTCTT GTTTACTTTT ACCAGGAGAT GCCACTGTCA TAACTTCTTC 120 ATGGGATAAT AATGTCTATT TTTATTCCAT AGCATTTGGA AGACGCCAGG ACACGTTAAT 180

GGGACATGAT GATGCTGTTA GTAAGATCTG TTGGCATGAC AACAGGCTAT ATTCTGCATC 240

GTGGGACTCT ACAGTGAAGG TGTGGTCTGG TGTTCCTGCA GAGATGCCAG GCACCAAAAG 300

ACACCACTTT GACTTGCTGG CCGAGCTGGA ACATGATGTC AGTGTAGATA CAATCAGTTT 360

AAATGCTGCA AGCACACTGT TAGTTTCCGG CACCAAAGAA GGCACAGTGA ATATTTGGGA 420 CCTCACAACG GCC 433

(2) INFORMATION FOR SEQ ID NO: 2 :

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 140 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

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

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

20 25 30 lie Thr Ser Ser Trp Asp Asn Asn Val Tyr Phe Tyr Ser He Ala Phe 35 40 45

Gly Arg Arg Gin Asp Thr Leu Met Gly His Asp Asp Ala Val Ser Lys 50 55 60

He Cys Trp His Asp Asn Arg Leu Tyr Ser Ala Ser Trp Asp Ser Thr 65 70 75 80

Val Lys Val Trp Ser Gly Val Pro Ala Glu Met Pro Gly Thr Lys Arg 85 90 95

His His Phe Asp Leu Leu Ala Glu Leu Glu His Asp Val Ser Val Asp

100 105 110

Thr He Ser Leu Asn Ala Ala Ser Thr Leu Leu Val Ser Gly Thr Lys 115 120 125

Glu Gly Thr Val Asn He Trp Asp Leu Thr Thr Ala 130 135 140

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TTTTTTTNAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 60

AAAAAAAAAA AAAAAAAAAA AAAA 84

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 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:4 : AGTGTAGATA CAATCAGTTT AAATGCTGCA 30

(2) INFORMATION FOR SEQ ID NO:5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 254 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

AAGCTTTCCC TGCCAGGACA CAGCACTGTT TTCTAAAGGG AACAGATATT AGAGATGAGG 60

ACAGTATGGT TGGATTTGAA TATGATTGCT TAGATTGAAT AATTTCTCCA ATTGGAGTCT 120

GTTTTTCTCT AAGTTTTAAA TTGAGGANAA AACAGATGAG CCAGACATGG AAATTTCTAA 180

TTTTAAATTC AAATTTTGAA TATTTTGATT TAGTAATTTG AGAACAAGCA ATTTGTAATT 240

CTAGTGAGCC TTAC 254 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 733 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

;ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTCGACCTTG AAGATGTTTT CTAAAGAATC AAAAATGCTA CAAAGAAGTA TATCATTTTC 60

AAATATGGCT TTATCGTCTT GTTTACTTTT ACCAGGAGAT GCCACTGTCA TAACTTCTTC 120

ATGGGATAAT AATGTCTATT TTTATTCCAT AGCATTTGGA AGACGCCAGG ACACGTTAAT 180

GGGACATGAT GATGCTGTTA GTAAGATCTG TTGGCATGAC AACAGGCTAT ATTCTGCATC 240

GTGGGACTCT ACAGTGAAGG TGTGGTCTGG TGTTCCTGCA GAGATGCCAG GCACCAAAAG 300 ACACCACTTT GACTTGCTGG CCGAGCTGGA ACATGATGTC AGTGTAGATA CAATCAGTTT 360

AAATGCTGCA AGCACACTGT TAGTTTCCGG CACCAAAGAA GGCACAGTGA ATATTTGGGA 420

CCTCACAACG GCCACCTTAA TGCACCAGAT TCCATGCCAT TCAGGGATTG TATGTGACAC 480

TGCTTTTAGC CCAGATAGTC GCCATGTCCT CAGCACAGGA ACAGATGGCT GTCTTAATGT 540

CATTGATGTG CAGACAGGAA TGCTCATCTC CTCCATGACA TCAGATGAGC CCCAGAGGTG 600 CTTTGTCTGG GATGGAAATT CCGTTTTATC TGGCAGTCAG TCTGGTGAAC TGCTCGTTTG 660

GGACCTCCTT GGAGCAAAAA TCAGTGAGAG AATACAGGGC CACACAGGTG CTGTGACATG 720

TATATGGATG AAT 733

( 2 ) INFORMATION FOR SEQ ID NO : 7 :

( i ) SEQUENCE CHARACTERISTICS :

( A ) LENGTH : 240 amino ac ids

( B ) TYPE : amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :

Met Phe Ser Lys Glu Ser Lys Met Leu Gin Arg Ser He Ser Phe Ser 1 5 10 15

Asn Met Ala Leu Ser Ser Cys Leu Leu Leu Pro Gly Asp Ala Thr Val 20 25 30

He Thr Ser Ser Trp Asp Asn Asn Val Tyr Phe Tyr Ser He Ala Phe 35 40 45 Gly Arg Arg Gin Asp Thr Leu Met Gly His Asp Asp Ala Val Ser Lys 50 55 60

He Cys Trp His Asp Asn Arg Leu Tyr Ser Ala Ser Trp Asp Ser Thr 65 70 75 80

Val Lys Val Trp Ser Gly Val Pro Ala Glu Met Pro Gly Thr Lys Arg 85 90 95

His His Phe Asp Leu Leu Ala Glu Leu Glu His Asp Val Ser Val Asp 100 105 110

Thr He Ser Leu Asn Ala Ala Ser Thr Leu Leu Val Ser Gly Thr Lys 115 120 125

Glu Gly Thr Val Asn He Trp Asp Leu Thr Thr Ala Thr Leu Met His 130 135 140

Gin He Pro Cys His Ser Gly He Val Cys Asp Thr Ala Phe Ser Pro 145 150 155 160

Asp Ser Arg His Val Leu Ser Thr Gly Thr Asp Gly Cys Leu Asn Val 165 170 175

He Asp Val Gin Thr Gly Met Leu He Ser Ser Met Thr Ser Asp Glu 180 185 190

Pro Gin Arg Cys Phe Val Trp Asp Gly Asn Ser Val Leu Ser Gly Ser 195 200 205 Gin Ser Gly Glu Leu Leu Val Trp Asp Leu Leu Gly Ala Lys He Ser

210 215 220

Glu Arg He Gin Gly His Thr Gly Ala Val Thr Cys He Trp Met Asn

225 230 235 240

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 296 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :

ACTTGTCAGA AAGCACTGAA CAATTAAGAA ATTTTCCAAG AAAATGTGCA GNATTCTCTG 60

CNACTTCAGA ATCTGTTTTG TCTTCCTAAT CNATCACAAT TGCCACCCAT CGGGTNTTGG 120

GTGTGTGTTT TCATAGCGTG GTTACTTTCT ATAATGCTGN ACCCAGATTC TAAGAACCTG 180

GAGAAGGATT AGCAGTTCTT AGTAAGTTTA CTGTGTATAG GAACGGTTTG TATTTCATTA 240 CAGCTATTCA TCTTTTCTAC ATTAAAAATA TTTTTCTCTA AAAAAAAAAA AAAAAA 296