AU2800101A - DNA sequences and secreted proteins encoded thereby - Google Patents

Description

AUSTRALIA

Patents Act 1990 Genetics Institute, Inc

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: DNA sequences and secreted proteins encoded thereby The following statement is a full description of this invention including the best method of performing it known to us:- 1A DNA SEQUENCES AND SECRETED PROTEINS ENCODED THEREBY This is a divisional of AU 67685/96, the entire contents of which are incorporated herein by reference.

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 cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning 0 techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered factor partial DNA/amino acid sequence of the factor in the case of hybridization cloning: activity of the factor 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 f 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 factors 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 PCRbased techniques. It is to these factors and the polynucleotides encoding them that the present invention is directed.

SUMMARY

In a first aspect, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 67 to nucleotide 348.

In a second aspect, the present invention provides an isolated polynucleotide encoding a fragment of the protein having the amino acid sequence of SEQ ID NO:8, the fragment having immunogenic activity.

In a third aspect, the present invention provides an isolated polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8.

In a fourth aspect, the present invention provides an isolated polynucleotide which is an allelic variant of SEQ ID NO:7.

In a fifth aspect, the present invention provides an isolated 15 polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 75 to nucleotide 356.

In a sixth aspect, the present invention an isolated polynucleotide encoding a fragment of the protein having the amino acid sequence of SEQ ID NO:10, the fragment having immunogenic activity.

20 In a seventh aspect, the present invention provides an isolated polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID In an eighth aspect, the present invention provides an isolated polynucleotide which is an allelic variant of SEQ ID NO:9.

25 In a ninth aspect, the present invention provides an isolated polynucleotide capable of hybridizing under stringent conditions to the polynucleotide according to any one of the first to eighth aspects, or to a complement thereof.

In a tenth aspect, the present invention provides a polynucleotide of the present invention operably linked to an expression control sequence.

In an eleventh aspect, the present invention provides a host cell transformed with a polynucleotide of the present invention.

In a twelfth aspect, the present invention provides a process for producing a polypeptide encoded by a polynucleotide of the present invention, which comprises: growing the host cell of the present invention in a suitable culture medium; and purifying the protein from the culture.

In a thirteenth aspect, the present invention provides a polypeptide produced according to a process of the present invention.

In a fourteenth aspect, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:8, said polypeptide being substantially free from other mammalian proteins.

In a fifteenth aspect, the present invention provides an isolated fragment of the polypeptide having the amino acid sequence of SEQ ID NO:8, said fragment having immunogenic activity.

In a sixteenth aspect, the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO:10, said polypeptide being S. substantially free from other mammalian proteins.

15 In a seventeenth aspect, the present invention provides an isolated fragment of the polypeptide having the amino acid sequence of SEQ ID NO:10, said fragment having immunogenic activity.

In an eighteenth aspect, the present invention provides a composition comprising a polypeptide of the present invention further comprising a 20 pharmaceutically acceptable carrier.

In a nineteenth aspect, the present invention provides a composition comprising a fragment of the present invention further comprising a pharmaceutically acceptable carrier.

In a twentieth aspect, the present invention provides a composition 25 comprising an antibody which specifically reacts with a polypeptide of the present invention.

In a twenty-first aspect, the present invention provides a composition comprising an antibody which specifically reacts with a fragment of the present invention.

In a twenty-second aspect, the present invention provides a method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effecting amount of a composition of the present invention.

In another aspect, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 52 to nucleotide 2034; a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3 encoding a protein having biological activity; a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity; a polynucleotide which is an allelic variant of SEQ ID NO:4; and a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in In another embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 76 to nucleotide 474; a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5 encoding a protein having biological activity; a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6; 20 a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity; a polynucleotide which is an allelic variant of SEQ ID NO:5; and a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in 25 In another aspect, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 86 to nucleotide 544; a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11 encoding a protein having biological activity; a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12; a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity; a polynucleotide which is an allelic variant of SEQ ID NO:11; and a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in 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: growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and purifying the protein from the culture.

The protein produced according to such methods is also provided by the present invention.

*00 o* g0 0 Compositions comprising a protein biological activity are also disclosed. In preferred embodiments the protein comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; fragments of the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:4; fragments of the amino acid sequence of SEQ ID NO:4; the amino acid sequence of SEQ ID NO:6; fragments of the amino acid sequence of SEQ ID NO:6; the amino acid sequence of SEQ ID NO:8; fragments of the amino acid sequence of SEQ ID NO:8; the amino acid sequence of SEQ ID NO:12; and fragments of the amino acid sequence of SEQ ID NO:12; the protein being substantially free from other mammalian proteins.

.*go 15 Such compositions 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 20 therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not 25 the exclusion of any other element, integer or step, or group of elements, integers or steps.

DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES The sequence of a polynucleotide encoding one protein of the present invention is set forth in SEQ ID NO:1, with the coding region extending from nucleotides 38 to 1447. This polynucleotide has been identified as "clone J5" The amino acid sequence of the protein encoded by clone J5 is set forth in SEQ ID NO:2.

Clone J5 was deposited with the American Type Culture Collection on August 11, 1995 and given the accession number ATCC 69885. SEQ ID NO:1 represents a spliced combination of sequence obtained from an isolated clone identified as "J5_3_fl", with additional 5' sequence obtained from a second double stranded clone.

Clone J5 was isolated from a human activated peripheral blood mononuclear cell (PBMC) library using a trap which selects for nucleotides encoding secreted proteins; therefore, clone J5 does encode a secreted factor. J5 encodes a novel protein; BLASTN/BLASTX or FASTA searches revealed no exact sequence matches.

20 However, a BLASTX search revealed homology between the J5 protein (in the approximate region of amino acids 62-129 of SEQ ID NO:2), epididymal apical proteins (including without limitation, epididymal apical protein I-precursor (Macaca fascicularis) (accession X66139)) and several snake venom haemorrhagic peptides (disintegrins) (including without limitation those assigned accession U01235-1237, 25 X68251, and M89784). Analysis of the full-length J5 sequences revealed that the "00 disintegrin domain was incomplete and that this clone did not contain an EGF-domain, 6 as with some of the other disintegrin family members. J5 does contain a conserved metallo-proteinase domain. Based upon these homologies, J5 and these homologous proteins are expected to share at least some activities.

The sequence of a polynucleotide encoding another protein of the present invention is set forth in SEQ ID NO:3, with the coding region extending from nucleotides 52 to 2034. This polynucleotide has been identified as "clone J422" The amino acid sequence of the protein encoded by clone J422 is set forth in SEQ ID NO:4. Clone J422 was deposited with the American Type Culture Collection on August 11, 1995 and given the accession number ATCC 69884. SEQ ID NO:3 represents a spliced combination of sequence obtained from an isolated clone identified as "J422_fl", with additional 5' sequence obtained from a second double stranded clone. Clone J422 was isolated from a human activated peripheral blood mononuclear cell (PBMC) library using a trap which selects for nucleotides encoding secreted proteins; therefore, clone J422 does encode a secreted factor. J422 encodes a novel protein; BLASTN/BLASTX or FASTA searches revealed no exact sequence matches. However, a FASTA search revealed homology between the J422 protein (in the approximate region of amino acids 34-156 of SEQ ID NO:4) and a number of Drosophila leucine-rich repeat (LRR) proteins. Analysis of the full-length J422 sequences revealed that the conserved EGF-domain found in a number of LRR family members was not present in J422. Based upon these homologies, J422 and these homologous proteins are expected to share at least some activities.

The sequence of a polynucleotide encoding another protein of the present invention is set forth in SEQ ID NO:5, with the coding region extending from nucleotides 76 to 474. This polynucleotide has been identified as "clone L105" The amino acid sequence of the protein encoded by clone L105 is set forth in SEQ ID ee. 20 NO:6. Clone L105 was deposited with the American Type Culture Collection on August 11, 1995 and given the accession number ATCC 69883. Clone L105 was isolated from a murine adult thymus library using a trap which selects for nucleotides encoding secreted proteins; therefore, clone L105 does encode a secreted factor. L105 encodes a novel protein; BLASTN/BLASTX or FASTA searches revealed no exact sequence matches. However, a BLASTX search revealed homology between the L105 protein (particularly in the approximate region of amino acids 73-91 of SEQ ID NO:6), various monocyte and other chemoattractant proteins (including without limitation those assigned accession M577441, X71087, X72308, X14768 and M24545) and a chicken (Gallus gallus) cytokine (accession L34553). Based upon these homologies, L105 and these homologous proteins are expected to share at least some activities.

The sequence of polynucleotides encoding another protein of the present invention is set forth in SEQ ID NO:7 and SEQ ID NO:9, with the coding regions extending from nucleotides 67 to 348 and nucleotides 75 to 356, respectively. These polynucleotides have been identified as "clone H174-10" and "clone H174-43", respectively (collectively referred to herein as "H174"). The amino acid sequence of 9 the protein encoded by clones H174 is set forth in SEQ ID NO:8 and SEQ ID Clone H174 was deposited with the American Type Culture Collection on August 11, 1995 and given the accession number ATCC 69882. Clones H174 were isolated from a human activated peripheral blood mononuclear cell (PBMC) library using a trap which selects for nucleotides encoding secreted proteins: therefore, H174 does encode a secreted factor. H174 encodes a novel protein: BLASTN/BLASTX or FASTA searches revealed no exact sequence matches. However, a BLASTX search revealed homology between the H174 protein, human IP-10 (accession M33266) and murine CRG-2 (accession M86820) (species homologs). Based upon these homologies, H174 and these homologous proteins are expected to share at least some activities.

The sequence of a polynucleotide encoding another protein of the present invention is set forth in SEQ ID NO:11, with the coding region extending from nucleotides 86 to 544. This polynucleotide has been identified as "B18" The amino acid sequence of the protein encoded by clone B18 is set forth in SEQ ID NO:12.

Clone B18 was deposited with the American Type Culture Collection on July 6, 1995 o and assigned accession number ATCC 69868. Clone B18 was isolated from a human activated peripheral blood mononuclear cell (PBMC) library using a trap which selects for nucleotides encoding secreted proteins; therefore, clone B18 does encode a secreted factor. B 18 encodes a novel protein; BLASTN/BLASTX or FASTA searches revealed no exact sequence matches. However, a BLASTX search revealed that the region from amino acid 29 to amino acid 163 of B18 (SEQ ID NO: 12) shows marked homology to portions of murine CTLA-8 (amino acids 18 to 150, accession L13839) and herpesvirus Saimiri ORF13 ("herpes CTLA-8") (amino acids 19 to 151, accession X64346). Based upon these homologies, B 18 is believed to be the human homolog of murine and herpes CTLA-8 "human CTLA-8"). B18 may demonstrate proinflammatory activity, particularly in development of T-cell dependent immune responses. B 18 is also expected to possess other activities specified herein.

Polynucleotides hybridizing to the polynucleotides of the present invention under stringent conditions and highly stringent conditions are also part of the present invention. As used herein, "highly stringent conditions" include, for example, at least about 0.2xSSC at 65 0 C; and "stringent conditions" include, for example, at least about 4xSSC at 65 0 C or at least about 50% formamide, 4xSSC at 42 0 C. Allelic variants of the polynucleotides of the present invention are also encompassed by the invention.

Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.

Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al.. Bio/Technology 10, 773- 778 (1992) and in R.S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing 20 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 25 of the invention.

The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the 0o protein recombinantly. 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 Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the 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 primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary 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, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.

Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the Sprotein produced therein, for example by phosphorylation or glycosylation of the Sappropriate sites, in order to obtain the functional protein. Such covalent attachments 20 may be accomplished using known chemical or enzymatic methods.

The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in 25 kit form from, Invitrogen, San Diego, California, U.S.A. (the MaxBac@ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the S•present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification 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, heparin-toyopearl® or 12 Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins 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 purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification 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 purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein." e The protein of the invention may also be expressed as a product of transgenic animals, as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized 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 sharing primary, secondary or tertiary structural and/or conformational characteristics 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, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

13 The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.

Mutagenic techniques for such replacement, insertion or deletion are well known to those skilled in the art (see, U.S. Patent No. 4,518,584).

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

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

S°RESEARCH TOOL UTILITY The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers (when labeled) to map related gene positions; to compare with endogenous 14 DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences: as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; to raise antiprotein antibodies using DNA immunization techniques: and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used to raise antibodies or to elicit another immune response; as a reagent (including the labelled reagent) in assays designed to quantitatively determine levels of the protein (or its o t receptor) in biological fluids; as markers for tissues in which the corresponding 20 protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors 25 of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

.Any or all of these "research tool" utilities are capable of being developed into reagent grade or kit format for commercialization as "research products." CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION

ACTIVITY

A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, B9, B9/11, BaF3, MC9/G, M+ (preB 2E8, RB5, DA1, 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 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.

20 Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, 25 Toronto. 1994; and Measurement of mouse and human Interferon y, 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.

SAssays 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, 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.

173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 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 11 Bennett, Giannotti, Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto.

1991; Measurement of mouse and human Interleukin 9 Ciarletta. Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.

*e USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai 20 et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

IMMUNE STIMULATING/SUPPRESSING

ACTIVITY

A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), 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 HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria. leshmania, malaria and various fungal infections such as candida. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally would be indicated, in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graftversus-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 or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, asthma and related respriatory conditions), may also be treatable using a protein of the present invention.

A protein of the present invention may also suppress chronic or acute inflammation, such as, for example, that associated with infection (such as septic S• shock or systemic inflammatory response syndrome (SIRS)), inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF •20 or IL-1 (such as the effect demonstrated by IL-11).

The activity of a protein of the invention may, among other means, be measured by the following methods: coos*: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, 25 A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing l* Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.

128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J.

Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992- 1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

18 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: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. 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.

*e eo* Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

20 Dendritic cell-dependent assays (which will identify, among others, proteins expressed by denritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimenal Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 25 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, S""i 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those 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 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 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 various 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 20 of myeloid cells such as granulocytes and monocytes/macrophages traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or Streat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia. and generally for use 0* 25 in place of or complimentarily to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility 0 in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo in conjunction with bone marrow transplantation) 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 embyronic differentation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece. I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.

20 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, 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 initating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, 25 Wiley-Liss, Inc., New York, NY. 1994.

TISSUE GENERATION/REGENERATION ACTIVITY *i 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 in the treatment of bums, 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.

21 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 20 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 25 tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or S.ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

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

•0o It is expected that a protein of the present invention may also exhibit activity o for generation of other tissues, such as organs (including, for example, pancreas, liver, 20 intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition of fibrotic to scarring to allow normal tissue to regenerate.

A protein of the present invention may also be useful for gut protection or 25 regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

The activity of a protein of the invention may, among other means, be S* measured by the following methods: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon); International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No. W091/07491 (skin, endothelium).

ACTIVIN/INHIBIN ACTIVITY A protein of the present invention may also exhbit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.

Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-P group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.

A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.

The activity of a protein of the invention may, among other means, be S measured by the following methods: 20 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.

25 CHEMOTACTIC/CHEMOKINETIC ACTIVITY A protein of the present invention may have chemotactic or chemokinetic activity act as a chemokine) for mammalian cells, including, for example, monocytes, neutrophils, T-cells, mast cells, eosinophils and/or endothelial cells.

Chemotactic and chemokinetic proteins can be used to mobilized or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

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

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those 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-Intersciece (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller 20 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 25 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 of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction or stroke).

The activity of a protein of the invention may, among other means, be measured by the following methods: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

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

The activity of a protein of the invention may, among other means, be measured by the following methods: 0 20 Suitable assays for receptor-ligand activity 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-Intersciece (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, 25 1987; Biereret al., J. Exp. Med. 168:1145-1156. 1988; Rosenstein et al., J. Exp.

0* Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

OTHER ACTIVITIES A protein of the invention may also exhibit one or more of the following additional activities or effects: killing infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin or other tissue pigmentation, or organ size (such as, for example, breast augmentation or diminution); effecting the processing of dietary fat, protein or carbohydrate; effecting behavioral characteristics, 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 ebryonic stem cells in lineages other than hematopoietic lineages; and in the case of enzymes, correcting deficiencies of the enzyme and treating related diseases.

ADMINISTRATION AND DOSING A protein of the present invention (from whatever source derived, 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 carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that •does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of 20 administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, 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 25 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 heterodimers or 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 multimeric or complexed form.

The pharmaceutical composition of the invention may be in the form of a complex of the protein(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 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 antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical compositon of the invention.

The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other 20 pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within 25 the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028: and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

28 In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(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 cytokine(s), lymphokine(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 20 a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, 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, 25 capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about to 95% protein of the present invention, and preferably from about 25 to protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.

When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.

A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has 20 undergone. Ultimately, the attending physician will decide the amount of protein of S"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 S•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 25 that point the dosage is not increased further. It is contemplated that the various 000.

pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 lpg to about 100 mg (preferably about 0. lpg to about 10 mg, more preferably about 0.1 pg 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. 211, 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 20 antibodies against the protein may be useful in detecting and preventing the metastatic o.

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 25 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 proteincontaining composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being 31 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 welldefined, 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, 20 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 S* 25 compositions from disassociating from the matrix.

o* A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, 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 32 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-a and TGF-P), and insulin-like growth factor (IGF).

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

The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, amount of tissue weight .desired to be formed, the site of damage, the condition of the damaged tissue, the size S of a wound, type of damaged tissue bone), the patient's age, sex, and diet, the 20 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 to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of 25 tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for gene therapy.

:Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).

Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

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

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

a I

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C C o 34 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Jacobs, Kenneth McCoy, John Kelleher, Kerry Carlin, McKeough (ii) TITLE OF INVENTION: DNA SEQUENCES AND SECRETED PROTEINS ENCODED THEREBY (iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Genetics Institute, Inc. Legal Affairs STREET: 87 CambridgePark Drive CITY: Cambridge STATE: Massachusetts COUNTRY: USA ZIP: 02140 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: 0" NAME: Brown, Scott A.

REGISTRATION NUMBER: 32,724 REFERENCE/DOCKET NUMBER: GI6000 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (617) 498-8224 TELEFAX: (617) 876-5851 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2209 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 38..1447 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAGAAGATAA AACTGGACAC TGGGGAGACA CAACTTC ATG CTG CGT GGG ATC TCC Met Leu Arg Gly Ile Ser 1 CAG CTA CCT GCA GTG GCC ACC ATG Gin Leu Pro Ala Val Ala Thr Met TGG GTC CTG CTG Trp Val Leu Leu CCT GTA CTT Pro Val Leu 103 151 TGG CTC ATT Trp Leu Ile GTT CAA ACT CAA Val Gin Thr Gin

GCA

Ala 30 ATA GCC ATA AAG CAA ACA CCT GAA Ile Ala Ile Lys Gin Thr Pro Glu TTA ACG Leu Thr CTC CAT GAA ATA GTT TGT CCT AAA AAA Leu His Glu Ile Val Cys Pro Lys Lys 45

CTT

Leu CAC ATT TTA CAC His Ile Leu His

AAA

Lys AGA GAG ATC AAG Arg Glu Ile Lys AAC CAG ACA GAA Asn Gin Thr Glu CAT GGC AAA GAG His Gly Lys Glu AGG TAT GAA CCT Arg Tyr Glu Pro

GAA

Glu GTT CAA TAT CAG Val Gin Tyr Gin ATC TTA AAT GGA Ile Leu Asn Gly GAA GAA Glu Glu ATC ATT CTC Ile Ile Leu ACT GAA ACA Thr Glu Thr 105 CTA CAA AAA ACC Leu Gin Lys Thr CAC CTC. CTG GGG His Leu Leu Gly CCA GAC TAC Pro Asp Tyr 100 ACG AAA CCT Thr Lys Pro TTG TAC TCA CCC Leu Tyr Ser Pro

AGA

Arg 110 GGA GAG GAA ATT Gly Glu Glu Ile GAG AAC Glu Asn 120 ATG GAA CAC TGT Met Glu His Cys

TAC

Tyr 125 TAT AAA GGA AAC Tyr Lys Gly Asn

ATC

Ile 130 CTA AAT GAA AAG Leu Asn Glu Lys

AAT

Asn 135 TCT GTT GCC AGC Ser Val Ala Ser AGT ACT TGT GAC Ser Thr Cys Asp

GGG

Gly 145 TTG AGA GGA TAC Leu Arg Gly Tyr

TTC

Phe 150 343 391 439 487 535 583, 631 679 ACA CAT CAT CAC CAA AGA TAC CAG ATA Thr His His His Gin Arg Tyr Gin Ile 155 CCT CTG AAA AGC Pro Leu Lys Ser ACA GAC Thr Asp 165 GAG AAA GAA Glu Lys Glu GCT AAC CAC Ala Asn His 185

CAT

His 170 GCC GTC TTT ACA Ala Val Phe Thr

TCT

Ser 175 AAC CAG GAG GAA Asn Gin Glu Glu CAA GAC CCA Gin Asp Pro 180 CAA GGC CCA Gin Gly Pro ACA TGT GGT GTG Thr Cys Gly Val

AAG

Lys 190 AGC ACT GAC GGG Ser Thr Asp Gly ATT CGA Ile Arg 200 ATC TCT AGA TCA Ile Ser Arg Ser AAA AGC CCA GAG Lys Ser Pro Glu

AAA

Lys 210 GAA GAC TTT CTT Glu Asp Phe Leu

CGG

Arg 215 GCA CAG AAA TAC Ala Gin Lys Tyr GAT CTC TAT TTG Asp Leu Tyr Leu

GTG

Val 225 CTG GAT AAT GCC TTT Leu Asp Asn Ala Phe 230 TAT AAG AAC TAT AAT GAG AAT CTA ACT CTG ATA AGA AGC TTT GTG TTT 36 Tyr Lys Asn Tyr Asn Glu Asn Leu Thr Leu Ile Arg Ser Phe Val Phe 235 240 245 see.

O* 0 0 0000 0 0000 0 0 00*0 0

GAT

Asp

GTG

Val

GTG

Val

AGT

Ser 295

GGG

Gly

TTG

Leu

AAT

Asn

GGT

Gly

GTG

Val 375

TCT

Ser

TGC

Cys

TGT

Cys

CCT

Pro

AAG

GTG

Val.

GCC

Ala

GTG

Val.

280

TCT

Ser

ATT

Ile

TGT

Cys

GTG

Val1

ATG

Met 360

ATG

Met

TGC

Cys

CTG

Leu

GGG

Giy

AAG

Lys 440

CCT

ATG

Met

TTG

Leu 265

CCC

Pro

AAC

Asn

AGC

Ser

TCC

Ser

GCT

Ala 345

CCT

Pro

AAT

Asn

CGT

Arg

CTG

Leu

AAC

Asn 425

GAG

Giu

GGA

Asn 250

GTA

Val

AGC

Ser

CTG

Leu

TTC

Phe

CCA

Pro 330

CTT

Leu

GAT

Asp

CAG

Gin

GCA

Ala

CAA

Gin 410

CAC

His

TGT

Cys

ACT

Leu

GGT

Gly

GCA

Ala

GGG

Giy

AAC

Asn 315

TCT

Ser

GTA

Val1

GTT

Val

TAT

Tyr

CAT

His 395

GCA

Ala

CTT

Leu

ACC

Thr

GAT

Leu Asn ATG GAA Met Giu AGC ACC Ser Thr 285 AAA AAG Lys Lys 300 AAT CGA Asn Arg TCG GTT Ser Val GGA GTG Gly Val CCA TTC Pro Phe 365 CTG AGT Leu Ser 380 TTT GAA Phe Giu CCT ATT Pro Ile CTA GAA Leu Giu AAT CTC Asn Leu 445 TGC GGA la 1

W.C

Ile 270 'kCG rhr

PATC

Ile

CGT

Arg

GCT

Ala

ATG

Met 350

AAC

Asn

TCA

Ser

AGA

Arg

CCT

Pro

GTG

Val.

430

TGC

Cys

GG?

Ile 255

TGG

Trp

TTT

Phe

CAC

His

GTG

Val

GTT

Val 335

TCA

Ser

ACC

Thr

AAA

Lys

TAC

Tyr

*ACA

Thr 415

GGA

*Giy

TGT

Cys

GAT

Tyr

TCT

Ser

GAC

Asp

GAC

Asp

GGA

Gly 320

ATT

Ile

CAT

His

AAG

Lys

TTC

Phe

CTT

Leu 400

AAT

Asn

GAA

Giu

GAA

Giu

GCT

Asn

GAT

Asp

AAC

As n

CAT

His 305

CTG

Leu

GAG

Glu

GAG

Giu

TGT

Cys

CCA

Pro 385

TTA

Leu

ATA

Ile

GAC

Asp

GCC

Ala

CCA

Thr

~GGG

Gly

TTC

Phe 290

GCT

Ala

GCA

Ala

GCT

Ala

CTG

Leu

CCC

Pro 370

AAG

Lys

TCT

Ser

ATG

Met

TGT

Cys

CTA

Leu 450

AAC

Ile

GAT

Asp 275

CTG

Leu

CAG

Gin

GCT

Ala

AAA

Lys

GGC

Gly 355

TCT

Ser

GAT

Asp

CAG

Gin

ACA

Thr

GAT

Asp 435

ACG

Thr

CAT

Asp 260

AAG

Lys

AGA

Arg

CTT

Leu

TCA

Ser

AAA

Lys 340

CAT

His

GGC

Gly

TTC

Phe

AAA

Lys

ACA

Thr 420

TGT

Cys

TGI

Cys

ACC

Vail

ATA

Ile

TGG

Trp

CTC

Leu

AAT

Asn 325

AAG

Lys

GTC

Val

AGT

Ser

AGT

Ser

*CCA

Pro 405

CCA

Pro

GGC

*Giy

~AAA

*Lys

*ACA

AIAG

Lys

CAC

His

AGC

Ser 310

TCC

Ser

AAT

Asn

CTT

Leu

TGT

Cys

ACA

Thr 390

AAG

Lys

GTG

Val.

TCT

Ser

CTG

Leu

GAG

AAC CTA CTC AAT GTG ATA TAT AAC ACC ATA GAT GTT CAA 823 871 919 967 1015 1063 liii 1159 1207 1255 1303 1.351 1399 1447 1507 1567 1627 1687 Lys Pro Giy Thr Asp 455 Gly Gly Asp Ala Pro Asn His Thr Thr Glu TGAATCCAAA AGTCTGCTTC ACTGAGATGC TACCTTGCCA AACTGTCCCA GGAATCTTGT GAATTTTCAC CCATAATGGT

GGACAAGAAC

CTTTCACTTG

CAAGAACTCT

TCATTCTACT

TTCTATATTG TTATCAGTCC AGGAAACAGG TAAACAGATG TAATTAGAGA CATTGGCTCT TTGTTTAGGC CTAATCTTTC TTTTTACTTT TTTTTTTCTT TTTTCTTTTT TTTTAAAGAT

CATGAATTTG

TCACCTTAAA

AGTACTATGC

ACTTGATGGG

CCTCTGTGTT

ATGATTCAAT

TCATCATATA

AAAGAAGGCT

TGACTTAGTT

ATGCACGGCT

TTTAATGCTT

TGAAGGCTTT

CACTTCGCCT

GTAAATATTT

TGAAGTTTAT

TAATGATTTA

CTGCCCTTTG

AAACTATTCA

CTTTCATCTT

GGCAATAGAA

TGCTCTTGAA

TTCATTTTAT

TTTGAGAAGT

TTGAACCATA

GAGAACAAAA

GAGTTAACAC

ACTAGTATGG

AGAAGAATAG

AGTGCAGTAT

CATGTATATC

CTACATTGCT

ATGTCAATAA

GAAAGCAGTC

TCCAGAATTG

CCTATAAAAA

AATTCAGGTT

TTTTCTACAT

CTATACACAC

TACATTTTAA

AAACACAACT

AA

TTCCATCAAA

TTAAATTACA

AAATAATACC

TTATGTTAT'r

CATGTCGAGA

ATCTCCTTCA.

TTGAGCCAGC

TTTGAGGCAA

1747 1807 1867 1927 1987 2047 2107 2167 2209 INFORMATION FOR SEQ ID NO:2: Ci) SEQUENCE CHARACTERISTICS: CA) LENGTH: 470 amino acids TYPE: amino acid TOPOLOGY: linear 000.

0 0 (ii) (xi) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu Arg Gly Leu Leu Pro 20 Lys Gin Thr Ser Gin Leu Pro Val Val. Ala Thr Met Ser Trp Val Leu Trp Leu Ile 25 Leu Gin Thr Gin Ile Pro Giu Leu 35 Lys Leu His 50 Thr His Glu Ile Ala Ile Ala Cys Pro Lys Gin Thr Glu Ile Leu His Lys Arg Giu Ile Lys Ile His Gly Lys Glu Giu 70 A.rg Tyr Giu Pro Giu 75 Val Gin Tyr Gin Met Leu Asri Gly Giu Ile Ile Leu Leu Gin Lys Thr Lys His Leu Leu Gly Giu Ile Thr 115 Asn Ile Leu 130 Tyr Thr Giu Thr 105 Met Leu Tyr Ser Pro Lys Pro Giu Asn 120 Ser Giu His Cys Arg Gly Giu 110 Tyr Lys Gly Thr Cys Asp Asn Giu Lys Asn 135 Thr Val Ala Ser Gly 145 Pro Leu Arg Giy Tyr Phe 150 His His His Gin 155 Ala Tyr Gin Ile Lys 160 Asn Leu Lys Ser Asp Glu Lys Giu Val Phe Thr Ser 175 Gin Giu Giu Gin Asp Pro Ala Asn His Thr Cys Giy Val Lys Ser Thr 180 185 190 Poo ae* .0 Do .5 600: *5 Asp Giu Val 225 Ile Asn Asp Asn His 305 Leu Giu Giu Cys Pro 385 Leu Ile Asp Ala Pro 465 Giy Lys 210 Leu Arg Thr Giy Phe 290 Ala Ala Ala Leu Pro 370 Lys Ser Met Cys Leu 450 Asn Lys 195 Giu Asp Ser Ile Asp 275 Leu Gin Ala Lys Gly 355 Ser Asp Gin Thr Asp 435 Thr His Gin Asp Asn Phe Asp 260 Lys Arg Leu Ser Lys 340 His Gly Phe Lys Thr 420 Cys Cys Thr Gly Pro Phe Leu Ala Phe 230 Val Phe 245 Val Gin Ile Lys Trp His Leu Ser 310 Asn Ser 325 Lys Asn Val Leu Ser Cys Ser Thr 390 Pro Lys 405 Pro Val Gly Ser Lys Leu Thr Giu 470 Ile Arg 215 Tyr Asp Val Val1 Ser 295 Gly Leu Asn Giy Val 375 Ser Cys Cys Pro Lys 455 Arg 200 Ala Lys Val1 Ala Val1 280 Ser Ile Cys Val Met 360 Met Cys Leu Gly Lys 440 Ilie Gin Asn Met Leu 265 Pro Asn Ser Ser Ala 345 Pro Asn Arg Leu Asn 425 Glu Ser Lys Tyr Asn 250 Val Ser Leu Phe Pro 330 Leu Asp Gin Ala Gin 410 His Cys Arg Tyr Asn 235 Leu Gly Ala Giy Asn 315 Ser Val Val1 Tyr His 395 Ala Leu Thr Ser Ile 220 Giu Leu Met Ser Lys 300 Asn Ser Gly Pro Leu 380 Phe Pro Leu Asn Cys 460 Leu 205 Asp Asn Asn Glu Thr 285 Lys Arg Val1 Val1 Phe 365 Ser Giu Ile Giu Leu 445 Lys Leu Leu Val Ile 270 Thr Ile Arg Al a Met 350 Asn Ser Arg Pro Val1 430 Cys Ser Tyr Thr Ile 255 Trp Phe His Val1 Val 335 Ser Thr Lys Tyr Thr 415 G ly Cys Pro Leu Leu 240 Tyr Ser Asp Asp Gly 320 Ile His Lys Phe Leu 400 Asn Giu Glu Pro Gly Thr Asp Gly Gly Asp Ala INFORMATION FOR SEQ ID NO:3: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 2582 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 52. .2034 (xi) SEQUENCE DESCRIPTION: SEQ ID, NO: 3: ATTTCTCAGC TCCAAGCATT AGGTAAACCC ACCAAGCAAT CCTAGCCTGT G ATG GCG Met Ala

S

S.

S

S.

S. S S S

S.

555555

S

S

C S

S

S

SSS

S

TTT

Phe

AAA

Lys

AAA

Lys 35

CTA

Leu

ACA

Thr

GAT

Asp

AGC

Ser

TTC

Phe 115

TTC

Phe

AAT

Asn

TCC

Ser

GAT

GAC

Asp

GTC

Val

ACA

Thr

CCA

Pro

ATT

Ile

TTA

Leu

CAT

His 100

ATG

Met

TTA

Leu

CTG

Leu

ATT

Ile

TTT

AGC

Ser

ACC

Thr

AAC

Asn

ACA

Thr

AAT

Asn 70

AGG

Arg

CAA

Gin

GAA

Giu

CAA

Gin

AAC

Asn 150

TTC

Phe

AAT

TGC

Cys

TCC

Ser

TGT

Cys

ACA

Thr

AGA

Arg

TGC

CYS

TTA

Leu

ACA

Thr

ACG

Thr 135

TTG

Leu

CCC

Pro

AAT

TTC

Phe

TGG

Trp

GAA

Giu 40

GAA

Giu

ACC

Thr

CAG

Gln

AGC

Ser

TCG

Ser 120

GGA

Gly

GAA

Giu

AAA

Lys

GCT

TTT

Phe

GAT

Asp 25

AAT

Asn

TTT

Phe

TTC

Phe

ATT

Ile

ACA

Thr 105

CTT

Leu

ATA

Ile

AGC

Ser

GAC

Asp

ATA

TGG

Trp

CAG

Gin

TTA

Leu

TTG

Leu

AGC

Ser

AAC

Asn

CTT

Leu

AAT

Asn

TCC

Ser

TTG

Leu

TTC

Phe 170

CAC

GTG

Val1

ATG

Met

GGT

Gly

GAA

Glu

AGA

Arg

TGG

Trp,

GTG

Val

GGG

Gly

AAT

Asn

TAT

Tyr 155

CCA

Pro

TAC

TTT

Phe

GAG

Giu

GAA

Giu

TTT

Phe

AAT

Asn

GAA

Giu

GGA

Gly 110

TCA

Ser

TTT

Phe

TGT

Cys

AAC

Asn

ACT

Thr

CCT

Pro

TTG

Leu

CAA

Gin

ATA

Ile

CTT

Leu 130

CAC

His

TCC

Ser

CTG

Leu

AGG

AGC AAC CAT Ser Asn His 160 AAT CTG AAA Asn Leu Lys 175 AGA GAA GAC 585 633 SI Asp

TCT

Ser 195

GTT

Val1

TTG

Leu

CAG

Gin

GAT

Asp

TCT

Ser 275

TCC

Ser

GCA

Ala

CTC

Leu

ATC

Ile

AAC

Asn 355

AAC

Asn

TGC

Cys

CTG

Leu Phe 180

CTG

Leu

AAA

Lys

AAC

Asn

AAC

Asn

GAC

Asp 260

GTT

Vai

ACC

Thr

ACT

Thr

AAG

Lys

AGT

Ser 340

GTG

Val

CTT

Leu

TGC

Cys

AGC

Ser Gin Asn GAG CAG Giu Gin GGT ATT Giy Ile TTT GGA Phe Giy 230 TCT ACT Ser Thr 245 GAA GAT Giu Asp GAG AGC Giu Ser ACA TTT Thr Phe CAC TTG H is Leu 310 AAA TTA Lys Leu 325 GCT GCC Ala Ala AAG AAA Lys Lys CAG ACA Gin Thr AGT CTG Ser Leu 390 CAC AAT His Asn 405 Asn

GCC

Ala

GAG

Giu 215

GGA

Giy

ACT

Thr

ATT

Ile

CTC

Leu

CAG

Gin 295

AAA

Lys

GTT

Val

AAT

Asn Ala

ATC

Ile 200

CTT

Leu

ACT

Thr

CAG

Gin

AGT

Ser

AAC

Asn 280

TGC

Cys

GGG

Gly

CTC

Leu

TTC

Phe Ile 185

AAC

Asn

GGG

Gly

CCA

Pro

TCT

Ser

TCA

Ser 265

CTG

Leu

TTC

Phe

TTA

Leu

AGT

Ser

CCC

Pro 345

CTT

Leu

TTA

Leu

.AA

Lys

CTT

Leu His

CTA

Leu

GCT

Ala

AAT

Asn

CTC

Leu 250

GCC

Ala

CAG

Gin

ACC

Thr

CCC

Pro

GTA

Val 330

TCC

Ser

GGT

Giy

AGC

Ser

A.AC

Asn

GGT

G iy 410 Tyr

AGC

Ser

TTT

Phe

TTG

Leu 235

TGG

Trp

ATG

Met

GAA

Giu

CA.A

Gin

TCT

Ser 315

AAT

Asn

CTT

Leu

GTT

Val

CAT

His

CTG

Leu 395

CTC

Leu Ile Ser Arg 190 CTG AAC TTC Leu Asn Phe 205 GAT TCA ACG Asp Ser Thr 220 TCT GTT ATA Ser Vai Ile CTG GGA ACA Leu Gly Thr CTC AAG, GGA Leu Lys Giy 270 CAC CGC TTC His Arg Phe 285 CTC CAA GAA Leu Gin Giu 300 GGG ATG AAG Gly Met Lys CAT TTC GAT His Phe Asp ACA CAC CTC Thr His Leu 350 GGC TGC TTG Gly Cys Leu 365 AAT GAC ATA Asn Asp Ile 380 TCC CAC TTG Ser His Leu CAG AGT CAG Gin Ser Gin

AAT

Asn

GTC

Val

TTC

Phe

TTT

Phe 255

CTC

Leu

TCT

Ser

TTG

Leu

GGT

Gly

CAA

Gin 335

TAC

Tyr

GAG

Glu

GAG

Giu

CAA

Gin

GCA

Ala 415

GGC

G iy

TTC

Phe

AAT

Asn 240

GAG

Giu

TGT

Cys

GAC

Asp

GAT

Asp

CTG

Leu 320

TTG

Leu

ATC

Ile

AA

Lys

GCT

Al a

ACC

Thr 400

TTC

Phe

AAT

Asn

CAA

Gin 225

GGT

Gly

GAC

Asp

GAA

Giu

ATC

Ile

CTG

Leu 305

AAC

Asn

TGT

Cys

AGA

Arg

CTA

Leu

TCT

Ser 385

TTA

Leu

AAA

Lys

AAT

Asn 210

AGT

Ser

CTG

Leu

ATT

Ile

ATG

Met

TCA

Ser 290

ACA

Thr

TTG

Leu

CAA

Gin

GGC

Giy

GGA

Gly 370

GAC

Asp

AAC

Asn

GAA

Giu 681 729 777 825 873 921 969 1017 1065 1113 1161 1209 1257 1305 1353 Giu Asp Met Arg CTT CAC Leu His 360 CTT GAT Leu Asp 375 CAA CTC Gin Leu GAG CCT Giu Pro TGT CCT CAG CTA GAA CTC CTC GAT TTG GCA TTT ACC CGC TTA CAC ATT Cys Pro Gin Leu Giu Leu Leu Asp Leu Ala Phe Thr Arg Leu His Ile 420 425 430 42 CAA AAC CTC AAT GCT CCA CAA AGT CCC TTC CAT TTC CTT CAG GTT Asn Ala Pro Gin Ser Pro Phe Gin Asn Leu His Phe Leu Gin Val

AAT

As n

GGC

Gly

GAT

Asp

GAG

Glu

GCA

Ala 515

AGC

Ser

TAC

Tyr

CTC

Leu

CTG

Leu

AAC

Asn 595

CCA

Pro

ATT

Ile

GCT

Ala

CAA

CTC ACT Leu Thr CTA CCA Leu Pro GGG ACT Gly Thr 485 GTT CTG Val Leu 500 TTC CAC Phe His CTG ACA Leu Thr CTC AAT Leu Asn CCT ATC Pro Ile 565 GAC TGC Asp Cys 580 CTG CAC Leu His TCT CTA Ser Leu ACA GCC Thr Al a ATT CTG Ile Leu 645 CAC ATT

TAC

Tyr

GTT

Val 470

ATC

Ile

ATT

Ile

AGC

Ser

TGC

Cys

CTG

Leu 550

TTG

Leu

ACT

Thr

AAA

Lys

AGG

Arg

PITA

Ile 630

CTA

TGC

Cys 455

CTC

Leu

ACG

Thr

TTG

Leu

TTG

Leu

GAC

Asp 535

GCT

Ala

TCC

Ser

TGC

Cys

CTT

Leu

GGA

Giy 615

GGC

Gly

TTT

TTC

Phe

CGG

Arg

AAG

Lys

TCC

Ser

GGA

Giy 520

AGC

Ser

GCC

Ala

CAG

Gin

TCG

Ser

GAA

Giu 600

GTT

Vali

ATT

Ile

TTT

CTT GAT ACC Leu Asp Thr CAT CTC AAC His Leu Asn 475 ACC AAC CTA Thr Asn Leu 490 TCT TGT GGT Ser Cys Giy 505 AAA ATG AGC Lys Met Ser ATT GAT TCT Ile Asp Ser AAC AGC ATT Asn Ser Ile 555 CAG AGC ACC Gin Ser Thr 570 AAT ATT CAT Asn Ile His 585 GGC TCG GAG Giy Ser Giu AAG CTA TCT Lys Leu Ser TTC TTT CTC Phe Phe Leu 635 GCA GTT AAA

AGC

Ser 460

TTA

Leu

CTT

Leu

CTC

Leu

CAT

His

CTT

Leu 540

AAC

Asn

ATT

Ile

TTC

Phe

GAG

Giu

GAT

Asp 620

ATA

Ile 445

AAT

Asn

A.A

Lys

CAG

Gin

CTC

Leu

GTA

Vai 525

AGC

Ser

ATC

Ile

AAT

Asn

TTA

Leu

ACC

Thr 605

GTC

Val

GTA

Val

CAG

Gin

GGG

Giy

ACC

Thr

TCT

Ser 510

GAC

Asp

CAT

His

ATC

Ile

TTA

Leu

ACA

Thr 590

ACG

Thr

AAG

Lys

TTT

Phe

CAT

His

AAT

Asn

GTG

Val1 495

ATA

Ile

TTA

Leu

CTT

Leu

TCA

Ser

AGT

Ser 575

TGG

Trp

TGT

Cys

CTT

Leu

CTA

Leu CTT CTA Leu Leu 465 CAC TTT His Phe 480 GGC AGC Gly Ser GAC CAG Asp Gin AGC CAC Ser Hi-4s AAG GGA Lys Giy 545 CCC CGT Pro Arg 560 CAT AAC His Asn TAC AAA Tyr Lys GCA AAC Ala Asn TCC TGT Ser Cys 625 TTA TTG Leu Leu 640 TGG AAA Trp Lys

CTG

Leu 450

GCA

Ala

CAA

Gin

TTG

Leu

CAA

Gin

AAC

Asn 530

ATC

Ile

CTC

Leu

CCC

Pro

GAA

Glu

CCG

Pro 610

GGG

Gly

TTG

Leu

TAC

Tyr 1401 1449 1497 1545 1593 1641 1689 1737 1785 1833 1881 1929 1977 2025 2074 2134 2194 TAC CTT CTC AGG Leu Phe Phe Ala Vai Lys Tyr Leu Leu TAGTGCTGAA GGTTTCCAGA GAAAGCAAAT AAGTGTGCTT Gin His Ile 660 AGCAAAATTG CTCTAAGTGA AAGAACTGTC ATCTGCTGGT GACCAGACCA GACTTTTCAG ATTGCTTCCT GGAACTGGGC AGGGACTCAC TGTGCTTTTC TGAGCTTCTT ACTCCTGTGA GTCCCAGAGC

TAAAGAACCT

ATGCTGCTGT

GAGAGGCACA

GAGGGACTGG

GCAGGGACTG

ACTCACCGAC

ATCCCTCCCA

CACAATTTGT

CAGAGCTGAA

GTCTGGGTTC

TCAGTA.ATGT

TTATTTTAAA

TGAAAAAAAA

TCTAGGCAAG

GAGCCCTTTC

CCGGCCCCGG

GCACCACACA

GCCAGCCCAC

AGCCATTTGA

AAAAAAAA

TACACCGAAT

CGCATGTGGA

AGTCTCCCAC

CCCCGCCCCT

TACCCACCCC

GAAACTTAcT GACTCAGTCC AGAGGGTCAG AGAGTGGGAG

GAAGCAGAGG

AGGGAGGCCA

TTCCCCTTCT

GAAAGGAGAT

CATCAGCCCC

CACTACAGCA

TTGTGCTTGG

TGGGGACAAA

GTCTCAATCC

2254 2314 2374 2434 2494 2554 2582 INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 661 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ala Phe Asp Val Ser Cys Phe Phe Trp Val Val 1 5 Gly Cys Lys Val Ile Thr Ser Trp, Asp Gin Met Cys 20 Leu P'ne Ser Ala 15 Ile Giu Lys Giu Ala Asn Lys Thr Tyr Asn Cys Giu Asn Leu Gly Leu Ser Giu Ile P'ro 35 40 45 Asp Thr Leu Pro Asn Thr Thr Giu Phe Leu Giu Phe Ser Phe Asn Phe 50 55 Leu Pro Thr Ile His Asn Arg Thr Phe Ser Arg Leu Met Asn Leu Thr 65 70 75 Phe Leu Asp Leu Thr Arg Cys Gin Ile Asn Trp Ile His Giu Asp Thr 90 Phe Gin Ser His His Gin Leu Ser Thr Leu Val Leu Thr Gly Asn Pro 100 105 110 Leu Ile Phe Met Ala Glu Thr Ser Leu Asn Gly Pro Lys Ser Leu Lys 115 120 125 His Leu Phe Leu Ile Gin Thr Gly Ile Ser Asn Leu Giu Phe Ile Pro 130 135 140 Val His Asn Leu Giu Asn Leu Giu Ser Leu Tyr Leu Gly Ser Asn His 145 150 155 160 Ile Ser Ser Ile Lys Phe Pro Lys Asp Phe Pro Ala Arg Asn Leu Lys 165 170 175 Val Leu Asp Phe Gin Asn Asn Ala Ile His Tyr Ile Ser Arg Giu Asp 180 185 190 Met Arg Ser Leu Giu Gin Ala Ile Asn Leu Ser Leu Asn Phe Asn Giy a.

a Asn Gin 225 Giy Asp Giu Ile Leu 305 Asn Cys Arg Leu Ser 385 Leu Lys His Vali Leu 465 Phe Ser Gin Asn Vai 210 Ser Leu Leu Gin Ile Asp Met Ser 275 Ser Ser 290 Thr Aia Leu Leu Gin Ile Giy Asn 355 Giy Asn 370 Asp Cys Asn Leu Giu Cys Ile Asn 435 Leu Asn 450 Aia Giy Gin Asp Leu Giu Gin Aia Lys Asn Asn Asp 260 Vai Thr Thr Lys Ser 340 Vai Leu Cys Ser Pro 420 Aia Leu Leu Giy Vali 500 Phe Giy Phe Ser 245 Giu Giu Thr His Lys 325 Aia Lys Gin Ser Hi;s 405 Gin Pro Thr Pro Thr 485 Leu His Ile Giu 215 Giy Giy 230 Thr Thr Asp Ile Ser Leu Phe Gin 295 Leu Lys 310 Leu Val Aia Asn Lys Leu Thr Leu 375 Leu Gin 390 Asn Giu Leu Giu Gin Ser Tyr- Cys 455 Vai Leu 470 Ile Thr Ile Leu Ser Leu Leu Giy Thr Pro Gin Ser Ser Ser 265 Asn Leu 280 Cys Phe Giy Leu Leu Ser Phe Pro 345 His Leu 360 Asp Leu Leu Lys Pro Leu Leu Leu 425 Pro Phe 440 Phe Leu Ar g His Lys Thr Ser Ser 505 Giy Lys Ala Asn Leu 250 Aia Gin Thr Pro Vai 330 Ser Giy Ser Asn Giy 410 Asp Gin Asp Leu Asn 490 Cys Met Phe Leu 235 Trp Met Giu Gin.

Ser 315 Asn Leu Vai His Leu 395 Leu Leu Asn Thr Asn 475 Leu Giy Ser Asp 220 Ser Leu Leu His Leu 300 Giy His Thr G iy Asn 380 Ser Gin Aia Leu Ser 460 Leu Leu Leu His Ser Thr Vai Phe Vai Ile Phe Asn 240 Giy Thr Phe Giu 255 Lys Giy Leu Cys 270 Arg Phe Ser Asp 285 Gin Giu Leu Asp Met Lys Giy Leu 320 Phe Asp Gin Leu 335 His Leu Tyr Ile 350 Cys Leu Giu Lys 365 Aso Ile Giu Aia His Leu Gin Thr 400 Ser Gin Aia Phe 415 Phe Thr Arg Leu 430 His Phe Leu Gin 445 Asn Gin His Leu Lys Giy Asn His 480 Gin Thr Vai Giy 495 Leu Ser Ile Asp 510 Val Asp Leu Ser 525 515 520 His Asn Ser Leu Thr Cys Asp Ser Ile Asp Ser Leu Ser His Leu Lys 530 535 540 Gly Ile Tyr Leu Asn Leu Ala Ala Asn Ser Ile Asn Ile Ile Ser Pro 545 550 555 560 Arg Leu Leu Pro Ile Leu Ser Gin Gin Ser Thr Ile Asn Leu Ser His 565 570 575 Asn Pro Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu Thr Trp Tyr 580 585 590 Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu Glu Thr Thr Cys Ala 595 600 605 Asn Pro Pro Ser Leu Arg Gly Val Lys Leu Ser Asp Val Lys Leu Ser 610 615 620 Cys Gly Ile Thr Ala Ile Gly Ile Phe Phe Leu Ile Val Phe Leu Leu 625 630 635 640 Leu Leu Ala Ile Leu Leu Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp 645 650 655 Lys Tyr Gin His Ile 660 g INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 588 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA oo (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 76..474 (xi) SEQUENCE DESCRIPTION: SEQ ID CGGCCAAAGA GGCCTAAACT TGCGGCTGTC CATCTCACCT ACAGCTCTGG TCTCATCCTC AACTCAACCA CAATC ATG GCT CAG ATG ATG ACT CTG AGC CTC CTT AGC CTG 111 Met Ala Gin Met Met Thr Leu Ser Leu Leu Ser Leu 1 5 GTC CTG GCT CTC TGC ATC CCC TGG ACC CAA GGC AGT GAT GGA GGG GGT 159 Val Leu Ala Leu Cys Ile Pro Trp Thr Gin Gly Ser Asp Gly Gly Gly 20 CAG GAC TGC TGC CTT AAG TAC AGC CAG AAG AAA ATT CCC TAC AGT ATT 207 Gin Asp Cys Cys Leu Lys Tyr Ser Gin Lys Lys Ile Pro Tyr Ser Ile 35 GTC CGA GGC TAT AGG AAG CAA GAA CCA AGT TTA GGC TGT CCC ATC CCG 255 Val Arg Gly Tyr Arg Lys Gin Glu Pro Ser Leu Gly Cys Pro Ile Pro 50 55 46 GCA ATC CTG TTC TCA CCC CGG AAG CAC TCT AAG CCT GAG CTA TGT GCA 303 Ala Ile Leu Phe Ser Pro Arg Lys His Ser Lys Pro Glu Leu Cys Ala 70 AAC CCT GAG GAA GGC TGG GTG CAG AAC CTG ATG CGC CGC CTG GAC CAG 351 Asn Pro Glu Glu Gly Trp Val Gin Asn Leu Met Arg Arg Leu Asp Gin 85 CCT CCA GCC CCA GGG AAA CAA AGC CCC GGC TGC AGG AAG AAC CGG GGA 399 Pro Pro Ala Pro Gly Lys Gin Ser Pro Gly Cys Arg Lys Asn Arg Gly 100 105 ACC TCT AAG TCT GGA AAG AAA GGA AAG GGC TCC AAG GGC TGC AAG AGA 447 Thr Ser Lys Ser Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg 110 115 120 ACT GAA CAG ACA CAG CCC TCA AGA GGA TAGCCCAGTA GCCCGCCTGG 494 Thr Glu Gin Thr Gin Pro Ser Arg Gly 125 130 AGCCCAGGAG ATCCCCCACG AACTTCAAGC TGGGTGGTTC ACGGTCCAAC TCACAGGCAA 554 AGAGGGAGCT AGAAAACAGA CTCAGGAGCC GCTA 588 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ala Gin Met Met Thr Leu Ser Leu Leu Ser Leu Val Leu Ala Leu 5 10 Cys Ile Pro Trp Thr Gin Gly Ser Asp Gly Gly Gly Gin Asp Cys Cys 20 25 Leu Lys Tyr Ser Gin Lys Lys Ile Pro Tyr Ser Ile Val Arg Gly Tyr 40 Arg Lys Gin Glu Pro Ser Leu Gly Cys Pro Ile Pro Ala Ile Leu Phe 55 Ser Pro Arg Lys His Ser Lys Pro Glu Leu Cys Ala Asn Pro Glu Glu 70 75 Gly Trp Val Gin Asn Leu Met Arg Arg Leu Asp Gin Pro Pro Ala Pro 90 Gly Lys Gin Ser Pro Gly Cys Arg Lys Asn Arg Gly Thr Ser Lys Ser 100 105 110 Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg Thr Glu Gin Thr 115 120 125 Gin Pro Ser Arg Gly 130 47 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 966 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(ix) FEATURE: NAME/KEY: CDS LOCATION: 67..348 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTTCCAAGAA GAGCAGCAAA GCTGAAGTAG CAGCAACAGC ACCAGCAGCA

ACAGCAAAAA

ACAAAC ATG AOT GTG AAG GGC ATG GCT ATA GCC TTO GCT GTG ATA TTG Met Ser Val Lys Gly Met Ala Ile Ala Leu Ala Val Ile Leu 1 5 TGT GCT ACA GTT GTT CAA GGC TTC CCC ATG TTC AAA AGA GOA CGC TGT Cys Ala Thr Val Val Gln Gly Phe Pro Met Phe Lys Arg Oly Arg Cys 15 20 25 CTT TGC ATA GGC CCT GGG GTA AAA GCA GTG AAA GTG GCA GAT ATT GAG Leu Cys Ile Oly Pro Gly Val Lys Ala Val Lys Val Ala Asp Ile Glu 35 40 AAA GCC TCC ATA ATG TAC CCA AGT AAC AAC TOT GAC AAA ATA OAA GTG Lys Ala Ser Ile Met Tyr Pro Ser Asn Asn Cys Asp Lys Ile Glu Val 55 ATT ATT ACC CTG AAA GAA AAT AAA GGA CAA COA TOC CTA AAT CCC AAA Ile Ile Thr Leu Lys Glu Asn Lys Gly Oln Arg Cys Leu Asn Pro Lys 70 TCG AAO CAA OCA AGO CTT ATA ATC AAA AAA OTT GAA AGA AAO AAT TTT Ser Lys Gln Ala Arg Leu Ile Ile Lys Lys Val Glu Arg Lys Asn Phe so 85 TAAAAATATC AAAACATATG AAGTCCTOGA AAAGGGCATC TGAAAAACCT

AOAACAAGTT

TAACTGTGAC TACTGAAATG ACAAGAATTC TACAOTAGGA AACTGAGACT

TTTCTATGGT

TTTOTGACTT TCAACTTTTG TACAGTTATG TOAAGGATGA AAOGTGGGTG

AAAGGACCAA

AAACAGAAAT ACAGTCTTCC TGAATGAATG ACAATCAOAA TTCCACTGCC

CAAAGGAGTC

CAACAATTAA ATGGATTTCT AGGAAAAOCT ACCTTAAGAA AGGCTGGTTA

CCATCOOAGT

TTACAAAGTG CTTTCACOTT CTTACTTGTT GTATTATACA TTCATGCATT

TCTAGGCTAG

AGAACCTTCT AGATTTGATG CTTACAACTA TTCTGTTGTO ACTATGAGAA

CATTTCTGTC

TCTAGAAGTT ATCTGTCTGT ATTGATCTTT ATGCTATATT ACTATCTGTG

GTTACAGTGG

AGACATTOAC ATTATTACTO GAGTCAAGCC CTTATAAGTC AAAAGCACCT

ATGTGTCGTA

108 156 204 252 300 348 408 468 528 588 648 708 768 828 888 AAGCATTCCT CAAACATTTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 94 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Ser Val Lys Gly 5 TYPE: protein DESCRIPTION: SEQ ID Met Ala Ile Ala Leu 10 NO:8: Ala Val Met 1 Thr Ile Leu Cys Ala Val Val Gin Ile Gly Pro Gly Ser Ile Met Tyr Thr Leu Lys Glu Gly Phe Pro Met Phe Lys Arg 25 Val Lys Ala Val Lys Val Ala Pro Ser Asn Asn Cys Asp Lys 55 Asn Lys Gly Gin Arg Cys Leu 70 Asp Ile Gly Arg Cys Leu Cys Glu Lys Ala Val Ile Ile Ile Asn Pro Lys Ser 65 Gin Ala Arg Leu Ile Ile Lys Lys Val Glu Arg Lys Asn Phe INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1354 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 75..356 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTCTACTCCT TCCAAGAAGA GCAGCAAAGC TGAAGTAGCA AGCAAAAAAC AAAC ATG AGT GTG AAG GGC ATG GCT Met Ser Val Lys Gly Met Ala 1 5 ATA TTG TGT GCT ACA GTT GTT CAA GGC TTC CCC Ile Leu Cys Ala Thr Val Val Gin Gly Phe Pro 20 GCAACAGCAC CAGCAGCAAC ATA GCC TTG GCT GTG Ile Ala Leu Ala Val ATG TTC AAA AGA GGA Met Phe Lys Arg Gly CGC TGT CTT TGC ATA GGC CCT GGG GTA AAA GCA GTG AAA GTG GCA GAT Arg Cys Leu Cys Ile Gly Pro Giy Val Lys Ala Val Lys Val Ala Asp 35 ATT GAG AAA GCC TCC ATA ATG TAC CCA AGT AAC AAC TGT GAC AAA ATA Ile Glu Lys Ala Ser Ile Met Tyr Pro Ser Asn Asn Cys Asp Lys Ile 50 55 GAA GTG ATT ATT ACC CTG AAA GAA AAT AAA GGA CAA CGA TGC CTA A.AT Glu Val Ile Ile Thr Leu Lys Giu Asn Lys Gly Gin Arg Cys Leu Asn 70 CCC AAA TCG AAG CAA GCA AGG CTT ATA ATC AAA AAA GTT GAA AGA AAG Pro Lys Ser Lys Gin Ala Arg Leu Ile Ile Lys Lys Val Giu Arg Lys 85 AAT TTT TAAAAATATC AAAACATATG AAGTCCTGGA AAAGGGCATC TGAAAAACCT Asn Phe

AGAACAAGTT

TTTCTATGGT

AAAGGACCAA

CAAAGGAGTC

CCATCGGAGT

TCTAGGCTAG

CATTTCTGTC

GTTACAGTGG

ATGTGTCGTA

TATCATGTAG

AACCAATTCA

TACTGGCAAC

TTTTCAACTT

TTTTTGTTTT

AATGTTTTTG

CTTTTACTTT

TAACTGTGAC

TTTGTGACTT

AAACAGAAAT

CAACAATTAA

TTACAAAGTG

AGAACCTTCT

TCTAGAAGTT

AGACATTGAC

AAGCATTCCT

CACATCAATA

TTAAATGTAA

AGTGCACATA

TTATTCATTG

GATCCGTTTG

TCTACCAAAG

TTGTAATTCT

TACTGAAATG

TCAACTTTTG

ACAGTCTTCC

ATGGATTTCT

CTTTCACGTT

AGATTTGATG

ATCTGTCTGT

ATTATTACTG

CAAACATTTT

TGTAGGGAAA

TTCATAAAAT

TTTCATAACC

AGATGTTTTG

TATAAATGAT

AAAAATGTTG

GTCTCTTAGA

ACA.AGAATTC

TACAGTTATG

TGAATGAATG

AGGAAAAGCT

CTTACTTGTT

CTTACAACTA

ATTGATCTTT

GAGTCAAGCC

TTCATGCAAA

CATTCTTATG

GTACTATGAA

AAATTAGCAG

AAGCAATTAG

AGCAATATCT

AAAAATAAGC

AAAATACATA

TACAGTAGGA

TGAAGGATGA

ACAATCAGAA

ACCTTAAGAA

GTATTATACA

TTCTGTTGTG

ATGCTATATT

CTTATAAGTC

TACACACTTC

CATCATTTGG

AAAAATTATA

CACCGGTCTT

GATATGTGTG

TGGACACATT

AAATGTATAC

ATCTAATT

AACTGAGACT

AAGGTGGGTG

TTCCACTGCC

AGGCTGGTTA

TTCATGCATT

ACTATGAGAA

ACTATCTGTG

AAAAGCACCT

TTTCCCCAAA

TTTGTTTTAT

CGCTATGGGA

AATTTGATGT

TTTACTGTAC

TGAAATACAA

CTAGCA.ATCA

466 526 586 646 706 766 826 886 946 1006 1066 1126 1186 1246 1306 1354 INFORMATION FOR SEQ ID NO:iO: SEQUENCE CHARACTERISTICS: LENGTH: 94 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:iO: Met Ser Val Lys Gly Met Ala Ile Ala Leu Ala Val Ile Leu Cys Ala 1 5 10 Thr Val Val Gin Gly Phe Pro Met Phe Lys Arg Gly Arg Cys Leu Cys 25 Ile Gly Pro Gly Val Lys Ala Val Lys Val Ala Asp Ile Glu Lys Ala 40 Ser Ile Met Tyr Pro Ser Asn Asn Cys Asp Lys Ile Glu Val Ile Ile 55 Thr Leu Lys Glu Asn Lys Gly Gin Arg Cys Leu Asn Pro Lys Ser Lys 70 75 Gin Ala Arg Leu Ile Ile Lys Lys Val Glu Arg Lys Asn Phe INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 813 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

C

(ix) FEATURE: NAME/KEY: CDS LOCATION: 86..544 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGAAGATAC ATTCACAGAA AGAGCTTCCT GCACAAAGTA AGCCACCAGC GCAACATGAC AGTGAAGACC CTGCATGGCC CAGCC ATG GTC AAG TAC 1 ,TG CTG CTG TCG ATA 112 Met Val Lys Tyr Leu Leu Leu Ser Ile 1 TTG GGG CTT GCC TTT CTG AGT GAG GCG GCA GCT CGG AAA ATC CCC AAA 160 Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys Ile Pro Lys 15 20 GTA GGA CAT ACT TTT TTC CAA AAG CCT GAG AGT TGC CCG CCT GTG CCA 208 Val Gly His Thr Phe Phe Gin Lys Pro Glu Ser Cys Pro Pro Val Pro 35 GGA GGT AGT ATG AAG CTT GAC ATT GGC ATC ATC AAT GAA AAC CAG CGC 256 Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu Asn Gin Arg 50 GTT TCC ATG TCA CGT AAC ATC GAG AGC CGC TCC ACC TCC CCC TGG AAT 304 Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser Pro Trp Asn 65 TAC ACT GTC ACT TGG GAC CCC AAC CGG TAC CCC TCG GAA GTT GTA CAG 352 Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu Val Val Gin 80 51 GCC CAG TGT AGG AAC TTG GGC TGC ATC AAT GCT Ala Gin Cys Arg Asn Leu Gly Cys Ile Asn Ala 95 100 ATC TCC ATG AAT TCC GTT CCC ATC CAG CAA GAG Ile Ser Met Asn Ser Val Pro Ile Gin Gin Giu 110 115 AGG AAG CAC CAA GGC TGC TCT GTT TCT TTC CAG Arg Lys His Gin Giy Cys Ser Val Ser Phe Gin 125 130 GTG ACT GTT GGC TGC ACC TGC GTC ACC CCT GTC Val Thr Vai Gly Cys Thr Cys Val Thr Pro Val 140 145 TAAGAGGTGC ATATCCACTC AGCTGAAGAA GCTGTAGAAA TCTGCAACAA GTCCTGTCTG ACCCCCAATT CCCTCCACTT CTGCACGGAT GGAAACAGAA AATATTCACA ATGTATGTGT TTGATATCTA AAATGTTAGG AGAAAAATTA ATATATTCAG AATAATTTAA AAATAAAAAA AAAAAAAAA INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 153 amino acids TYPE: amino acid TOPOLOGY: linear CAA GGA AAG GAA GAC Gin Gly Lys Giu Asp ACC CTG GTC GTC CGG Thr Leu Val Val Arg 120 TTG GAG AAG GTG CTG Leu Giu Lys Val Leu 135 ATC CAC CAT GTG CAG Ile His His Val Gin 150 TGCCACTCCT TACCCAGTGC CACAGGACTC TTAATAAGAC GTATGTACTA CACTTTATAT TGCTAATATA ATAAAGTATT 400 448 496 544 604 664 724 784 813 (ii) MOLECULE TYPE: protein S

S

Met 1 Giu Lys Ile Giu Asn Cys Ile Val1 (xi) SEQUENCE Val Lys Tyr Leu 5 Ala Ala Aia Arg 20 Pro Giu Ser Cys 35 Gly Ile Ile Asn Ser Arg Ser Thr Arg Tyr Pro Ser Ilie Asn Ala Gin 100 Gin Gin Giu Thr 115 Ser Phe Gin Leu DESCRIPTION: SEQ ID Leu Leu Ser Ile Leu 10 Lys Ile Pro Lys Val 25 Pro Pro Val Pro Gly Giu Asn Gin Arg Val Ser Pro Trp Asn Tyr 70 Giu Val Val Gin Ala Gly Lys Giu Asp Ile 105 Leu Val Val Arg Arg 120 Giu Lys Val Leu Val NO: 12: Gly Leu Gly His Giy Ser Ser Met Thr Val 75 Gin Cys Ser Met Lys His Thr Val Phe Phe Lys Arg Trp Asn Ser 110 Gly Cys Leu Phe Leu As n Asp Leu Val Cys Thr Ser Gin Asp Ile Pro Gly Pro Ser Cys 130 135 140 Val Thr Pro Val Ile His His Val Gin 145 iSO t*0e 09...

Claims (26)

1. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 67 to nucleotide 348.

2. An isolated polynucleotide encoding a fragment of the protein having the amino acid sequence of SEQ ID NO:8, the fragment having immunogenic activity.

3. An isolated polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8.

4. An isolated polynucleotide which is an allelic variant of SEQ ID NO:7. An isolated polynucleotide capable of hybridizing under stringent conditions to the polynucleotide according to any one of claims 1 to 4, or to a complement thereof.

6. The polynucleotide according to any one of claims 1 to 5, wherein said polynucleotide is operably linked to an expression control sequence.

7. A host cell transformed with the polynucleotide according to any one of claims 1 to 6.

8. A process for producing a polypeptide encoded by the polynucleotide according to any one of claims 1 to 5, which comprises: growing the host cell of claim 7 in a suitable culture medium; and purifying the protein from the culture.

9. A polypeptide produced according to the process of claim 8. A polypeptide comprising the amino acid sequence of SEQ ID NO:8, said polypeptide being substantially free from other mammalian proteins.

11. An isolated fragment of the polypeptide having the amino acid sequence of SEQ ID NO:8, said fragment having immunogenic activity.

12. A composition comprising the polypeptide of claim 9 or claim further comprising a pharmaceutically acceptable carrier.

13. A composition comprising the fragment of claim 11 further comprising a pharmaceutically acceptable carrier.

14. A composition comprising an antibody which specifically reacts with the polypeptide of claim 9 or claim

15. A composition comprising an antibody which specifically reacts with the fragment of claim 11.

16. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a i therapeutically effecting amount of the composition of any one of claims 12 to

17. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 75 to nucleotide 356.

18. An isolated polynucleotide encoding a fragment of the protein "having the amino acid sequence of SEQ ID NO:10, the fragment having immunogenic activity.

19. An isolated polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID An isolated polynucleotide which is an allelic variant of SEQ ID NO:9.

21. An isolated polynucleotide capable of hybridizing under stringent conditions to the polynucleotide according to any one of claims 17 to 20, or to a complement thereof.

22. The polynucleotide according to any one of claims 17 to 21, wherein said polynucleotide is operably linked to an expression control sequence.

23. A host cell transformed with the polynucleotide according to any one of claims 17 to 22.

24. A process for producing a polypeptide encoded by the polynucleotide according to any one of claims 17 to 21, which comprises: growing the host cell of claim 23 in a suitable culture "medium; and purifying the protein from the culture. A polypeptide produced according to the process of claim 24. co' 26. A polypeptide comprising the amino acid sequence of SEQ ID said polypeptide being substantially free from other mammalian proteins.

27. An isolated fragment of the polypeptide having the amino acid sequence of SEQ ID NO:10, said fragment having immunogenic activity.

28. A composition comprising the polypeptide of claim 25 or claim 26 further comprising a pharmaceutically acceptable carrier.

29. A composition comprising the fragment of claim 27 further comprising a pharmaceutically acceptable carrier. A composition comprising an antibody which specifically reacts with the polypeptide of claim 25 or claim 26.

31. A composition comprising an antibody which specifically reacts with the fragment of claim 27.

32. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effecting amount of the composition of any one of claims 28 to 31. Dated this fourteenth day of March 2001 Genetics Institute, Inc. Patent Attorneys for the Applicant: *F B RICE CO *o ooo**