AU6053899A - Methods and compositions relating to egf-repeat-containing polypeptides - Google Patents

Description

WO00/17357 PCT/US99/21812 METHODS AND COMPOSITIONS RELATING TO EGF-REPEAT-CONTAINING POLYPEPTIDES 5 1. 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. 10 2. BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly 15 over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in 20 the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader 25 sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their -1- WO00/17357 PCT/US99/21812 secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention 5 is directed. 3. SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, in particular, novel EGF 10 repeat-containing polypeptides, isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotides, and antibodies that 15 specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such polypeptides. The compositions of the present invention additionally include vectors, including expression 20 vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides, and cells genetically engineered to express such polynucleotides. SEQ ID NO:1 represents a portion of the nucleotide 25 sequence encoding an EGF-repeat-containing polypeptide designated EGF-Hyl. The deduced amino acid sequence of SEQ ID NO:1 is set forth in SEQ ID NO:2. Further 5' sequence of EGF-Hyl was obtained and the resulting sequence is set forth in SEQ ID NO:10. The deduced amino 30 acid sequence of SEQ D NO:10 is set forth in SEQ ID NO:11. The polypeptide set out in SEQ ID NO:11 is 467 amino acids in length where amino acid 38 or other amino -2- WO00/17357 PCT/US99/21812 acid located further upstream may represent the initiating methionine. Three EGF repeat regions are located at about amino acid residues 349-385, 387-423, and 425-467 of SEQ ID NO:11 and are encoded by 5 nucleotides 1048-1158, 1162-1270, and 1276-1404 ,respectively, of SEQ ID NO:10 according to the Prosite program (http://www.expasy.ch/prosite/). Further cDNA or genomic DNA that corresponds to SEQ ID NO:1 or 10, including additional 5' cDNA sequences, can be obtained 10 by methods known in the art. For example, appropriate cDNA or genomic DNA libraries can be screened under suitable hybridization conditions using SEQ ID NO:1 or 10, or a portion thereof as a probe. Alternatively, SEQ ID NO:1 or 10 may be used as the basis for suitable 15 primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries. The true N-terminus of the mature protein can be confirmed by methods known in the art (e.g. through expression of the DNA in a mammalian or other host cell 20 that correctly processes the polypeptide. The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The isolated polynucleotides of the invention include, but 25 are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 11; a polynucleotide encoding a polypeptide comprising amino acid residues 1-14 of SEQ ID NO:2 (corresponding to amino acid residues 374-387 of SEQ ID 30 NO:11) ; a polynucleotide encoding a polypeptide comprising amino acid residues 15-54 of SEQ ID NO:2 (corresponding to amino acid residues 388-427 of SEQ ID -3- WO00/17357 PCT/US99/21812 NO:11); a polynucleotide encoding a polypeptide comprising amino acid residues 55-94 of SEQ ID NO:2 (corresponding to amino acid residues 428-467 of SEQ ID NO:11); or a polynucleotide encoding a polypeptide 5 comprising any one or more of the EGF repeat regions of SEQ ID NO:11. The isolated polynucleotides of the invention further include, but are not limited to a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 or 10; 10 a polynucleotide comprising nucleotides 3-284 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1123-1404 of SEQ ID NO:10); a polynucleotide comprising nucleotides 3-44 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1123-1164 of 15 SEQ ID NO:10); a polynucleotide comprising nucleotides 45-164 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1165-1284 of SEQ ID NO:10); a polynucleotide comprising nucleotides 165-284 of the nucleotide sequence of SEQ ID NO:1 (corresponding to 20 nucleotides 1285-1404 of SEQ ID NO:10); or a polynucleotide comprising any one or more of the EGF repeat coding regions of SEQ ID NO:10. The polynucleotides of the present invention still further include, but are not limited to, a polynucleotide 25 comprising the nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited on September 18, 1998 with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110, U.S.A., under 30 accession number ATCC 203198; a polynucleotide comprising the nucleotide sequence of the cDNA insert of clone pEGF HY1 deposited under accession number ATCC 203198; a -4- WO00/17357 PCT/US99/2 1812 polynucleotide comprising the full length protein coding sequence of a cDNA corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; a polynucleotide comprising the nucleotide 5 sequence of the mature protein coding sequence corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; or a polynucleotide encoding the mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under 10 accession number ATCC 203198. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes to the complement of the nucleotide sequence of SEQ ID NO:1 or 10 under stringent hybridization 15 conditions and that preferably has EGF-Hyl activity; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the proteins recited above; or a polynucleotide that encodes a 20 polypeptide comprising an EGF repeat region of a specific domain or truncation of the polypeptide of SEQ ID NO:1 or 10. The polynucleotides of the invention additionally include the complement of any of the polynucleotides 25 recited above. The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 11; a polypeptide comprising amino acid residues 1-14 of SEQ ID NO:2 30 (corresponding to amino acid residues 374-387 of SEQ ID NO:11); a polypeptide comprising amino acid residues 15 54 of SEQ ID NO:2 (corresponding to amino acid residues -5- WO00/17357 PCT/US99/21812 388-427 of SEQ ID NO:11); a polypeptide comprising amino acid residues 55-94 of SEQ ID NO:2 (corresponding to amino acid residues 428-467 of SEQ ID NO:11); or a polypeptide comprising any one or more of the EGF repeat 5 regions of SEQ ID NO:11. The polypeptides of the present invention further include, but are not limited to, a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 10 203198; a polypeptide comprising the full length protein encoded by a cDNA corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; a polypeptide comprising the mature protein corresponding to the cDNA insert of clone pEGF-HY1 15 deposited under accession number ATCC 203198; or a mature protein corresponding to the cDNA insert of clone pEGF HY1 deposited under accession number ATCC 203198. Preferred embodiments include those in which the protein produced by such process is a mature form of the 20 protein. Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e._g_., pharmaceutically acceptable, carrier. The invention also relates to methods for producing 25 a polypeptide comprising growing a culture of the cells of the invention in a suitable culture medium, and purifying the protein from the culture. Polynucleotides according to the invention have numerous applications in a variety of techniques known to 30 those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, -6- WO00/17357 PCT/US99/21812 use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or 5 tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization. In other exemplary embodiments, the polynucleotides 10 are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome. 15 The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the 20 polypeptide. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement. Methods are also provided for preventing, treating or ameliorating a medical condition which comprises 25 administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier. In particular, the polypeptides and polynucleotides 30 of the invention can be utilized, for example, as part of methods for stimulation of epithelial tissue growth, e.., skin regeneration. The polypeptides and -7- WO00/17357 PCT/US99/21812 polynucleotides of the invention may, therefore, be utilized, for example, as part of methods for tissue repair and regeneration, corneal transplant healing, burn treatment, skin graft production and administration, and 5 wound healing, e.__q., treatment of surgical incisions, and ulcers, such as stomach or diabetic ulcers. In addition, the polynucleotides and polypeptides of the invention can further be utilized, for example, as part of methods for the prevention and/or treatment of disorders involving 10 cell fate and differentiation, such as leukemias, as well as for the treatment of nervous disorders. The methods of the present invention further relate to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a 15 sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited above and for the identification of subjects exhibiting a predisposition to such conditions. Furthermore, the invention provides methods for 20 evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited herein. The EGF-Hyl protein of the present invention may be expressed in certain cancer cells, for example, brain, 25 lung, colon, gastrointestinal, breast, liver, prostate, kidney, urological, ovarian, endometrical, heart, bone tumors, blood cell malignancies such as leukemias, and skin cancers such as melanomas. Other EGF motif containing molecules have been previously linked to the 30 progression of various cancers. Expression of EGF-Hyl in tumor cells would indicate that this protein represents a potential marker of malignancy and a potential candidate -8- WO00/17357 PCTIUS99/21812 for small molecule therapeutic development for the treatment of certain tumors. Thus, the prognostic and diagnostic methods contemplated according to this aspect of the invention 5 include methods of detecting or quantitating EGF-Hyl polypeptides in tissue samples (e.g. biopsied tissue from brain, lung, or other tissues) or body fluid samples (e.g. cerebrospinal fluid, pleural fluid, sputum, ascites, blood, urine, or other fluids), particularly for 10 diagnosis, prognosis or monitoring of cancer. The invention also provides methods for the identification of compounds that modulate the expression of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, 15 for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. 20 The methods of the invention also include methods for the treatment of disorders as recited herein which may involve the administration of such compounds to individuals exhibiting symptoms or tendencies related to disorders as recited above. In addition, the invention 25 encompasses methods for treating diseases or disorders as recited herein by administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target 30 gene/protein expression or target protein activity. A linkage of EGF-Hyl with cancer would indicate that inhibitors of its activity (that either inhibit -9- WO00/17357 PCT/US99/21812 expression of the gene product or inhibit activity of the gene product itself) may be useful in treating cancer conditions. Such inhibitors include antisense polynucleotides, antibodies, and other modulators 5 identified thorough e.g. screening of libraries or combinatorial libraries of inorganic or organic compounds (such as bacterial, fungal, mammalian, insect or plant products, peptides, peptidomimetics and organomimetics). Such modulators may be administered parenterally, 10 including into the CSF, or locally via an implant or device. 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Top line: Nucleotide sequence of SEQ ID NO:1, 15 bottom line: amino acid sequence of SEQ ID NO:2. These sequences represent embodiments of the novel polynucleotides and polypeptides of the invention. Figure 2. EGF-repeat alignment. The amino acid sequence of SEQ ID NO:2 is aligned with consensus sequences of the 20 EGF-repeats of the Notch (SEQ ID NO:7), CD97 (SEQ ID NO:8), and EGF receptor (SEQ ID NO:9) proteins (labeled (C)). The Notch protein is involved in cell fate control mechanisms regulating multicellular development; CD97 is an activation-induced antigen on leukocytes which belongs 25 to a group of seven-span transmembrane (7-TM) molecules designated EGF-TM7; EGF is the epidermal growth factor. Gaps are presented as spaces and non-conserved residues as dashes. This alignment reveals that the amino acid sequence of SEQ ID NO:2 contains two and one half EGF 30 repeat domains, as indicated in the figure. Amino acid residue positions marking the beginning of each EGF repeat domain of SEQ ID NO:2 are provided. The single -10- WO00/17357 PCT/US99/21812 letter amino acid representation is used. In each of the sequences, conserved cysteine, glutamic acid and glycine residues are underlined. 5 5. DETAILED DESCRIPTION OF THE INVENTION 5.1. DEFINITIONS The term " nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms " nucleic acid" and 10 " polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of 15 oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene. 20 The terms "oligonucleotide fragment" or a "polynucleotide fragment", "portion," or "segment" is a stretch of polypeptide nucleotide residues which is long enough to use in polymerase chain reaction (PCR) or various hybridization procedures to identify or amplify 25 identical or related parts of mRNA or DNA molecules. The terms "oligonucleotides" or "nucleic acid probes" are prepared based on the polynucleotide sequences provided in the present invention. Oligonucleotides comprise portions of such a 30 polynucleotide sequence having at least about 15 nucleotides and usually at least about 20 nucleotides. Nucleic acid probes comprise portions of such a -11- WO00/17357 PCTUS99/21812 polynucleotide sequence having fewer nucleotides than about 6 kb, usually fewer than about 1 kb. After appropriate testing to eliminate false positives, these probes may, for example, be used to determine whether 5 specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P.S. et al., 1992, PCR Methods Appl 1:241-250). The term "probes" includes naturally occurring or 10 recombinant or chemically synthesized single- or double stranded nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are 15 elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, both of which are incorporated herein by 20 reference in their entirety. The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA under 25 in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 650 C, and washing in 0.1xSSC/0.1% SDS at 680 C), and moderately stringent conditions (i.e., washing in 0.2xSSC/0.1% SDS at 420 C). In instances wherein hybridization of 30 deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6xSSC/0.05% sodium pyrophosphate at 37 0 C (for 14-base -12- WO 00/17357 PCT/US99/21812 oligos), 48 0 C (for 17-base oligos), 55 0 C (for 20-base oligos), and 60 0 C (for 23-base oligos) The term "recombinant," when used herein to refer to a polypeptide or protein, means that a polypeptide or 5 protein is derived from recombinant (e.g., microbial or mammalian) expression systems. " Microbial" refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, " recombinant microbial" defines a polypeptide or protein 10 essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in 15 yeast will have a glycosylation pattern in general different from those expressed in mammalian cells. The term " recombinant expression vehicle or vector" refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An 20 expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and 25 translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by 30 a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue -13- WO00/17357 PCTIUS99/21812 may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product. The term "recombinant expression system" means host cells which have stably integrated a recombinant 5 transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA 10 segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers. Recombinant expression systems 15 as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic. 20 The term "open reading frame," ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein. The term " expression modulating fragment," EMF, 25 means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF. As used herein, a sequence is said to " modulate the expression of an operably linked sequence" when the expression of the sequence is altered by the presence of 30 the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments which induce -14- WO00/17357 PCT/US99/21812 the expression or an operably linked ORF in response to a specific regulatory factor or physiological event. As used herein, an " uptake modulating fragment," UMF, means a series of nucleotides which mediate the 5 uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed 10 by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of 15 uptake of a linked marker sequence. The term "active" refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide. The term "naturally occurring polypeptide" refers to 20 polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, 25 phosphorylation, lipidation and acylation. The term "derivative" refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionucleotides or various enzymes), pegylation (derivatization with polyethylene 30 glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins. -15- WO00/17357 PCT/US99/21812 The term "recombinant variant" refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. 5 Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, such as cellular trafficking, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the 10 number of amino acid sequence changes made in regions of high homology. Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical 15 properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar 20 (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) 25 amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by 30 systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule -16- WO00/17357 PCTIUS99/21812 using recombinant DNA techniques and assaying the resulting recombinant variants for activity. Alternatively, where alteration of function is desired, insertions, deletions or non-conservative 5 alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide 10 characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells 15 chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. As used herein, "substantially equivalent" can refer both to nucleotide and amino acid sequences, for example 20 a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially 25 equivalent sequence varies from one of those listed herein by no more than about 20% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided 30 by the total number of residues in the substantially equivalent sequence is about 0.2 or less). Such a sequence is said to have 80% sequence identity to the -17- WO00/17357 PCT/US99/21812 listed sequence. In one embodiment, a substantially equivalent, e.__., mutant, sequence of the invention varies from a listed sequence by no more than 10% (90% sequence identity); in a variation of this embodiment, by 5 no more than 5% (95% sequence identity); and in a further variation of this embodiment, by no more than 2% (98% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention generally have at least 95% sequence identity with a 10 listed amino acid sequence, whereas substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities (e.g. at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, or at least about 65%), 15 taking into account, for example, the redundancy or degeneracy of the genetic code. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered 20 substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Nucleic acid sequences encoding such substantially 25 equivalent sequences, e._g_., sequences of the recited percent identities, can routinely be isolated and identified via standard hybridization procedures well known to those of skill in the art. Where desired, an expression vector may be designed 30 to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the -18- WO00/17357 PCT/US99/21812 polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques. A polypeptide "fragment," "portion," or "segment" is 5 a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids. To be active, any polypeptide must have 10 sufficient length to display biologic and/or immunologic activity. Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic 15 code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide 20 sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. 25 The term "activated" cells as used in this application are those which are engaged in extracellular or intracellular membrane trafficking, including the export of neurosecretory or enzymatic molecules as part of a normal or disease process. 30 The term "purified" as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, -19- WO00/17357 PCT/US99/21812 e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99.8% by weight, of the indicated 5 biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present). The term "isolated" as used herein refers to a 10 nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) 15 only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms "isolated" and "purified" do not encompass nucleic acids or polypeptides present in their natural source. The term "infection" refers to the introduction of 20 nucleic acids into a suitable host cell by use of a virus or viral vector. The term "transformation" means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal 25 integration. The term "transfection" refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed. The term "intermediate fragment" means a nucleic 30 acid between 5 and 1000 bases in length, and preferably between 10 and 40 bp in length. -20- WO00/17357 PCTIUS99/21812 The term "secreted" protein refers to a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host 5 cell. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the 10 membrane of the endoplasmic reticulum. Each of the above terms is meant to encompasses all that is described for each, unless the context dictates otherwise. 15 POLYNUCLEOTIDES AND NUCLEIC ACIDS OF THE INVENTION Nucleotide and amino acid sequences of the invention are reported below. Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present 20 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 25 incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc 30 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 -21- WO00/17357 PCT/US99/21812 used to generate such fusions. For example, a protein- IgM fusion would generate a decavalent form of the protein of the invention. The present invention also provides both full-length 5 and mature forms of the disclosed proteins corresponding to SEQ ID NO:2 or 11. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein may 10 also be determinable from the amino acid sequence of the full-length form. The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with 15 known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic 20 materials. Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular 25 and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of 30 such domains from sequence information. Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. -22- WO00/17357 PCT/US99/21812 Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. 5 The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the 10 polynucleotides. The compositions of the present invention include isolated polynucleotides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic 15 variants, novel isolated polypeptides, and antibodies that specifically recognize one or more epitopes present on such polypeptides. Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. 20 Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of 25 the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides. 30 -23- WO 00/17357 PCTIUS99/21812 5.2. NUCLEIC ACIDS OF THE INVENTION The isolated polynucleotides of the invention include, but are not limited to, polynucleotides encoding a polypeptide comprising the amino acid sequence of SEQ 5 ID NO:2 or 11, polynucleotides encoding a full length or mature protein corresponding to SEQ ID NO:2 or 11, as well as to polynucleotides which encode EGF repeat regions or specific domains thereof. For example, the polynucleotides of the invention further include: a 10 polynucleotide encoding a polypeptide comprising amino acid residues 1-14 of SEQ ID NO:2 (corresponding to amino acid residues 374-387 of SEQ ID NO:11); a polynucleotide encoding a polypeptide comprising amino acid residues 15 54 of SEQ ID NO:2 (corresponding to amino acid residues 15 388-427 of SEQ ID NO:11) ; a polynucleotide encoding a polypeptide comprising amino acid residues 55-94 of SEQ ID NO:2 (corresponding to amino acid residues 428-467 of SEQ ID NO:11): or a polynucleotide encoding a polypeptide comprising any one or more of the EGF repeat regions of 20 SEQ ID NO:11. In particular embodiments, the isolated polynucleotides of the invention include, but are not limited to a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 or 10; a polynucleotide 25 comprising nucleotides 3-284 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1123-1404 of SEQ ID NO:10); a polynucleotide comprising nucleotides 3 44 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1123-1164 of SEQ ID NO:10); 30 a polynucleotide comprising nucleotides 45-164 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1165-1284 of SEQ ID NO:10); a polynucleotide -24- WO00/17357 PCT/US99/21812 comprising nucleotides 165-284 of the nucleotide sequence of SEQ ID NO:1 (corresponding to nucleotides 1285-1404 of SEQ ID NO:10); or a polynucleotide comprising any one or more of the EGF repeat coding regions of SEQ ID NO:10. 5 The polynucleotides of the present invention still further include, but are not limited to, a polynucleotide comprising the nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pEGF-HY1 deposited under accession number 10 ATCC 203198; a polynucleotide comprising the nucleotide sequence of the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; a polynucleotide comprising the full length protein coding sequence of a cDNA corresponding to the cDNA insert of clone pEGF-HY1 15 deposited under accession number ATCC 203198; a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; or a polynucleotide encoding the mature 20 protein corresponding to the cDNA insert of clone pEGF HY1 deposited under accession number ATCC 203198. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that preferably has EGF-Hyl biological activity and hybridizes 25 to the complement of the nucleotide sequence of SEQ ID NO:1 or 10 under stringent hybridization conditions; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the proteins 30 recited above; or a polynucleotide that encodes a polypeptide comprising an additional specific domain or truncation of the polypeptide of SEQ ID NO:2 or 11. -25- WO00/17357 PCT/US99/21812 The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotides of the invention also provide 5 polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence 10 identity to a polynucleotide recited above. The invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence 15 identity to a polynucleotide encoding a polypeptide recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for 20 example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities. A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences 25 by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e.g., plasmids, cosmids, lambda 30 phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the -26- WO00/17357 PCT/US99/21812 invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker 5 for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part 10 of a multicellular organism. The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic variations thereof. Allelic variations can be routinely 15 determined by comparing the sequence provided in SEQ ID NO:1 or 10, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 or 10, with a sequence from another isolate of the same species. Furthermore, to accommodate codon 20 variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another which encodes the same amino acid is 25 expressly contemplated. Any specific sequence disclosed herein can be readily screened for errors by resequencing a particular fragment, such as an ORF, in both directions (i.e., sequence both strands). The present invention further provides recombinant 30 constructs comprising a nucleic acid having the sequence of SEQ ID NO:1 or 10, or a fragment thereof. The recombinant constructs of the present invention comprise -27- WO00/17357 PCT/US99/21812 a vector, such as a plasmid or viral vector, into which a nucleic acid having the sequence of SEQ ID NO:1 or 10, or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of 5 the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. For vectors comprising the EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or 10 heterologous ORF operably linked to the EMF or UMF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are 15 provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNHl8a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, 20 pBPV, pMSG, pSVL (Pharmacia). 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 25 to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein 30 "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 -28- WO00/17357 PCT/US99/21812 protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence. Promoter regions can be selected from any desired 5 gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda Pz, and trc. Eukaryotic promoters include CMV 10 immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of 15 replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. 20 Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with 25 translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein 30 including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. -29- WO00/17357 PCT/US99/21812 Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in 5 operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic 10 hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. 15 As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 20 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These pBR322 " backbone" sections are combined with an appropriate promoter and the structural sequence to be 25 expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an 30 additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, -30- WO00/17357 PCT/US99/21812 and the resulting crude extract retained for further purification. Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequences 5 that hybridize under stringent conditions to a fragment of the DNA sequence in Figure 1 or its complement, which fragment is greater than about 10 bp, preferably 20-50 bp, and even greater than 100 bp. In accordance with the invention, polynucleotide sequences which encode the 10 novel nucleic acids, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. The nucleic acid sequences of the invention are 15 further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in 20 the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. The amino acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in 25 nature. These amino acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting 30 first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a -31- WO00/17357 PCT/US99/21812 charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically 5 contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 10 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells. In a preferred method, polynucleotides encoding the 15 novel nucleic acids are changed via site-directed mutagenesis. This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to 20 form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and 25 efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template 30 DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. -32- WO00/17357 PCT/US99/21812 PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the 5 corresponding region in the plasmid and this gives the desired amino acid variant. A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other 10 mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Bioloqv, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same 15 or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under 20 stringent conditions. 5.3. HOSTS The present invention further provides host cells genetically engineered to contain the polynucleotides of 25 the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the 30 polynucleotides of the invention, wherein such polynucleotides are in operative association with a -33- WO00/17357 PCT/US99/21812 regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, 5 such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et 10 al., Basic Methods in Molecular Biology (1986)). The host cells containing one of polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein 15 under the control of the EMF. Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well 20 as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian 25 cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with 30 prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, New York -34- WO00/17357 PCT/US99/21812 (1989), the disclosure of which is hereby incorporated by reference. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of 5 mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines. Mammalian expression vectors 10 will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the 15 SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial 20 extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid 25 chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. 30 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 -35- WO00/17357 PCT/US99/21812 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 5 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 10 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, 15 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 protein produced therein, for example by phosphorylation or glycosylation of the appropriate 20 sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods. In another embodiment of the present invention, cells and tissues may be engineered to express an 25 endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can 30 be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic -36- WO 00/17357 PCT/US99/21812 engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or 5 combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals. mRNA stability elements, splice 10 sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules. The targeting event may be a simple insertion of the 15 regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g__., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of 20 a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally 25 occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, 30 allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be -37- WO00/17357 PCT/US99/21812 facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively 5 selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes 10 Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene. The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent 15 No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (W093/09222) by Selden et al.; and International Application No. PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by 20 reference herein in its entirety. 5.4.POLYPEPTIDES OF THE INVENTION The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the 25 amino acid sequence of SEQ ID NO:2 or 11. The polypeptides of the invention further include polypeptides which comprise any one or more EGF repeat regions or specific domains of the amino acid sequence of SEQ ID NO:2 or 11, e.g., a polypeptide comprising amino 30 acid residues 1-14 of SEQ ID NO:2 (corresponding to amino acid residues 374-387 of SEQ ID NO:11) ; a polypeptide comprising amino acid residues 15-54 of SEQ ID NO:2 -38- WO00/17357 PCT/US99/21812 (corresponding to amino acid residues 388-427 of SEQ ID NO:11); a polypeptide comprising amino acid residues 55 94 of SEQ ID NO:2 (corresponding to amino acid residues 428-467 of SEQ ID NO:11); or a polypeptide comprising any 5 one or more of other EGF repeat regions identified herein. The polypeptides of the present invention further include, but are not limited to, a polypeptide comprising the amino acid sequence encoded by the cDNA insert of 10 clone pEGF-HY1 deposited under accession number ATCC 203198; a polypeptide comprising the full length protein encoded by a cDNA corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; a polypeptide comprising the mature protein 15 encoded by a cDNA corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198; or a mature protein corresponding to the cDNA insert of clone pEGF-HY1 deposited under accession number ATCC 203198. 20 Preferred embodiments include those in which the protein produced by such process is a mature form of the protein. Protein compositions of the present invention may further comprise an acceptable carrier, such as a 25 hydrophilic, e.g., pharmaceutically acceptable, carrier. The invention also relates to methods for producing a polypeptide comprising growing a culture of the cells of the invention in a suitable culture medium, and purifying the protein from the culture. For example, the 30 methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the -39- WO00/17357 PCT/US99/21812 invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, and further purified. 5 The invention further provides a polypeptide including an amino acid sequence that is substantially equivalent to SEQ ID NO:2 or 11. Polypeptides according to the invention can have at least about 95%, and more typically at least about 98%, sequence identity to SEQ ID 10 NO:2 or 11. The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By 15 " degenerate variant" is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid 20 fragments of the present invention are the ORFs that encode proteins. A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be 25 synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In an 30 alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can -40- WO00/17357 PCT/US99/21812 readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, 5 HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., 10 Current Protocols in Molecular Biology. The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to 15 express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for 20 introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention. The purified polypeptides can be used in in vitro binding 25 assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified 30 in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the -41- WO00/17357 PCT/US99/21812 molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells. In addition, the binding molecules may be complexed 5 with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to the tumor or other cell by the specificity of the binding molecule for SEQ ID NO:2 or 11. 10 The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. 15 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 20 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 25 natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies. The proteins provided herein also include proteins characterized by amino acid sequences similar to those of 30 purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences -42- WO00/17357 PCT/US99/21812 can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino 5 acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to 10 those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Other fragments and derivatives of the sequences of 15 proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be 20 encompassed by the present invention. 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. 25 Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods are well known in the art, as described in Summers and Smith, Texas 30 Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an -43- WO00/17357 PCT/US99/21812 insect cell capable of expressing a polynucleotide of the present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under culture conditions 5 suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The 10 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.R

M

. or Cibacrom blue 3GA Sepharose.R".; one or more steps involving 15 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 20 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 25 England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and In Vitrogen, 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 30 Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing -44- WO 00/17357 PCT/US99/21812 hydrophobic RP-HPLC media, e.g., 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, 5 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." 10 5.4. DEPOSIT OF CLONES The following clone, pEGF-HY1, was deposited with the American Type Culture Collection (ATCC) Manassas, Virginia, on September 18, 1998, and assigned the 15 indicated accession number. The clone represents a plasmid clone as described in the Examples set forth below. Microorganism/Clone ATCC Accession No. Clone pEGF-HY1 203198 20 5.5. USES AND BIOLOGICAL ACTIVITY The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses 25 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 -45- WO00/17357 PCT/US99/21812 proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). 5.5.1. RESEARCH USES AND UTILITIES 5 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 10 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 e.g. Southern gels; as chromosome markers or tags (when 15 labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR 20 primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression 25 patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for 30 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 -46- WO00/17357 PCT/US99/21812 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 5 similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively 10 determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); 15 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 20 identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Any or all of these research utilities are capable 25 of being developed into reagent grade or kit format for commercialization as research products. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation 30 "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in -47- WO00/17357 PCT/US99/21812 Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987. 5 5.5.2. NUTRITIONAL USES Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon 10 source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, 15 solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. 20 5.5.3. 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 25 inhibiting) activity or may induce production of other cytokines in certain cell populations. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in 30 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 -48- WO00/17357 PCT/US99/21812 present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 5 123, T1165, 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 10 in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiiey-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., 15 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., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994. 20 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. 25 Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin .gamma., 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. 30 Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and -49- WO 00/17357 PCTIUS99/21812 Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1 6.3.12, John Wiley and Sons, Toronto. 1991; deVries et 5 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 10 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11--Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 15 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9--Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. 20 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 25 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, 30 Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. -50- WO00/17357 PCT/US99/21812 Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988. 5.5.4. IMMUNE STIMULATING OR SUPPRESSING ACTIVITY 5 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 polynucleotide of the invention can encode a polypeptide exhibiting such 10 activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic 15 activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by vital (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or 20 other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the 25 present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. Autoimmune disorders which may be treated using a protein of the present invention include, for example, 30 connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, -51- WO00/17357 PCT/US99/21812 autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the 5 treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein 10 of the present invention. Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve 15 preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen 20 specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after 25 exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent. Down regulating or preventing one or more antigen 30 functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T -52- WO00/17357 PCT/US99/21812 cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue 5 transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B 10 lymphocyte antigen with its natural ligand(s) on immune cells prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this 15 matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance 20 by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens. 25 The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and 30 xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described -53- WO00/17357 PCT/US99/21812 in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 5 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease. Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. 10 Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may 15 reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived 20 cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating 25 autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen 30 arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., -54- WO00/17357 PCT/US99/21812 Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating 5 immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen 10 function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically. 15 Alternatively, anti-vital immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form 20 of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a 25 protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby 30 activate, T cells in vivo. The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the -55- WO00/17357 PCT/US99/21812 surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or 5 MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and P2 10 microglobulin protein or an MHC class II a chain protein and an MHC class II P chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B 15 lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also 20 be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be 25 sufficient to overcome tumor-specific tolerance in the subject. The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte 30 cytotoxicity 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. -56- WO00/17357 PCT/US99/21812 Strober, Pub. Greene Publishing Associates and Wiley Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. 5 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., I. 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, 10 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 15 133:327-341, 1991; Brown et al., J. Immunol. 153:3079 3092, 1994. Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody 20 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. 25 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 30 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 -57- WO00/17357 PCT/US99/21812 Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. 5 Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992. Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without 10 limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., 15 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, 1994; and Inaba et al., Journal of Experimental Medicine 20 172:631-640, 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, 25 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., 30 Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992. -58- WO00/17357 PCT/US99/21812 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 5 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991. 5.5.5. HEMATOPOIESIS REGULATING ACTIVITY A protein of the present invention may be useful in 10 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 15 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 20 erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat 25 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 in place of or complimentary to 30 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 -59- WO00/17357 PCTUS99/21812 hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal 5 hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal 10 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 15 above. Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: 20 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 25 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, N.Y. 1994; Hirayama 30 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. -60- WO00/17357 PCT/US99/21812 A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. 5 E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. 10 eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994. 15 5.5.6. TISSUE GROWTH ACTIVITY A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as 20 well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers. A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone 25 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 30 fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection -61- WO00/17357 PCT/US99/21812 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 5 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 10 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. 15 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 20 such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament like tissue inducing protein may have prophylactic use in 25 preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present 30 invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery -62- WO00/17357 PCTIUS99/21812 for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, 5 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 10 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 15 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 20 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 25 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 30 disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from -63- WO00/17357 PCT/US99/21812 chemotherapy or other medical therapies may also be treatable using a protein of the invention. Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, 5 including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. It is expected that a protein of the present invention may also exhibit activity for generation or 10 regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. 15 Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity. A protein of the present invention may also be 20 useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. A protein of the present invention may also be 25 useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above. The activity of a protein of the invention may, among other means, be measured by the following methods: 30 Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, -64- WO00/17357 PCT/US99/21812 tendon); International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium). Assays for wound healing activity include, without 5 limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978). 10 5.5.7. ACTIVIN/INHIBIN ACTIVITY A protein of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such 15 characteristics. 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 20 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 25 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 30 molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of the invention may also be useful -65- WO00/17357 PCT/US99/21812 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. 5 The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 10 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986. 5.5.8. CHEMOTACTIC/CHEMOKINETIC ACTIVITY 15 A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A 20 polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in 25 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. 30 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 -66- WO00/17357 PCTIUS99/21812 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 5 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 10 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 15 for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of a 20 and P Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140 146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994. 25 5.5.9. HEMOSTATIC AND THROMBOLYTIC ACTIVITY A protein of the invention may also exhibit hemostatic or thrombolytic activity. A polynucleotide of the invention can encode a polypeptide exhibiting such 30 attributes. Such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance -67- WO00/17357 PCT/US99/21812 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 5 treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke). The activity of a protein of the invention may, among other means, be measured by the following methods: 10 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 15 35:467-474, 1988. 5.5.10. RECEPTOR/LIGAND ACTIVITY A protein of the present invention may also demonstrate activity as receptors, receptor ligands or 20 inhibitors or agonists of receptor/ligand interactions. A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases 25 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 30 antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential -68- WO00/17357 PCT/US99/21812 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 5 inhibitors of receptor/ligand interactions. The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols 10 in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. 15 Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995. 20 5.5.11. ANTI-INFLAMMATORY ACTIVITY Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or 25 promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more 30 directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute -69- WO00/17357 PCT/US99/21812 conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, 5 arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat 10 anaphylaxis and hypersensitivity to an antigenic substance or material. 5.5.12. LEUKEMIAS Leukemias and related disorders may be treated or 15 prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic 20 leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott 25 Co., Philadelphia). 5.5.13. NERVOUS SYSTEM DISORDERS Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds 30 that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, -70- WO00/17357 PCTIUS99/21812 include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which 5 may be treated in a patient (including human and non human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: 10 (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; 15 (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; 20 (iii) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system 25 tissue; (iv) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by 30 human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; -71- WO00/17357 PCT/US99/21812 (v) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to 5 degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (vi) lesions associated with nutritional diseases or disorders, in which a portion 10 of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, 15 tobacco-alcohol amblyopia, Marchiafava Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vii) neurological lesions associated with 20 systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (viii) lesions caused by toxic substances 25 including alcohol, lead, or particular neurotoxins; and (ix) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but 30 not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or -72- WO00/17357 PCT/US99/21812 various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis. Therapeutics which are useful according to the 5 invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful 10 according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vi2vo; 15 (iii) increased production of a neuron associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or 20 (iv) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set 25 forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron 30 associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor -73- WO00/17357 PCT/US99/21812 neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability. 5 In a specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor 10 neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral 15 sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease). 20 5.5.14. OTHER ACTIVITIES A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or 25 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, fat to lean ratio or 30 other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting -74- WO00/17357 PCTIUS99/21812 biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, 5 lipid, protein, carbohydrate, vitamins, minerals, co factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including 10 depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, 15 correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and 20 the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein. 25 5.5.15 GENE THERAPY Mutations in the EGF-Hyl gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal EGF-Hyl activity or to treat disease states involving EGF-Hyl 30 (for example, various forms of cancer described herein). Delivery of a functional EGF-Hyl gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of -75- WO00/17357 PCT/US99/21812 vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, 5 Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275- 1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it 10 is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of EGF-Hyl will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of 15 EGF-Hyl. 5.5.16 TRANSGENIC ANIMALS In methods to determine biological functions of EGF Hyl in vivo, one or more EGF-repeat-containing genes are 20 either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic 25 animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non human mammals, can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein by reference. 30 Transgenic animals are useful to determine the role(s) EGF-Hyl play in biological processes, and preferably in disease states. Transgenic animals are useful as model -76- WO00/17357 PCT/US99/21812 systems to identify compounds that modulate EGF-Hyl activity. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Patent No 5,489,743 and PCT Publication No. WO94/28122, 5 incorporated herein by reference. Transgenic animals can be prepared wherein all or part of an EGF-Hyl promoter is either activated or inactivated to alter the level of expression of the EGF Hyl protein. Inactivation can be carried out using 10 homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more 15 heterologous enhancer elements known to confer promoter activation in a particular tissue. Knowledge of EGH-Hyl DNA sequences allows for modification of cells to permit, or increase, expression of endogenous EGF-Hyl. Cells can be modified (e.g., by 20 homologous recombination) to provide EGF-Hyl expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells EGF-Hyl protein at higher levels. The heterologous promoter is inserted in such a 25 manner that it is operatively linked to EGF-Hyl encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. It is also contemplated 30 that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate -77- WO00/17357 PCT/US99/21812 synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the EGF-Hyl coding sequence, amplification of the marker DNA by 5 standard selection methods results in co-amplification of the EGF-Hyl coding sequences in the cells. 5.6. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION 10 A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be administered to a patient in need, by itself, or in 15 pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, 20 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 25 route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL 9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, 30 TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its -78- WO 00/17357 PCT/US99/21812 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. 5 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 10 hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions 15 of the invention may comprise a protein of the invention in such multimeric or complexed form. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing 20 Co., Easton, PA, latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an 25 increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically 30 effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, -79- WO00/17357 PCT/US99/21812 whether administered in combination, serially or simultaneously. In practicing the method of treatment or use of the present invention, a therapeutically effective amount of 5 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 10 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 15 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), 20 other hematopoietic factor(s), thrombolytic or anti thrombotic factors. 5.6.1. ROUTES OF ADMINISTRATION Suitable routes of administration may, for example, 25 include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular 30 injections. Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be -80- WO00/17357 PCT/US99/2 1812 carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the 5 patient is preferred. Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or 10 sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery 15 system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue. 20 5.6.2. COMPOSITIONS/FORMULATIONS Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries 25 which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, 30 dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. -81- WO00/17357 PCT/US99/21812 When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in 5 tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of 10 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 15 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 20 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 25 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 30 pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle -82- WO 00/17357 PCT/US99/21812 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 5 the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible 10 buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. 15 For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, 20 liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding 25 suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, 30 potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone -83- WO00/17357 PCT/US99/21812 (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For 5 this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may 10 be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as 15 soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, 20 optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should 25 be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently 30 delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, -84- WO00/17357 PCT/US99/21812 trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules 5 and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or 10 continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and 15 may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, 20 suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous 25 injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of 30 the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a -85- WO00/17357 PCT/US99/21812 suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, 5 e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for 10 example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly 15 soluble derivatives, for example, as a sparingly soluble salt. A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a 20 water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute 25 ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co 30 solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components -86- WO00/17357 PCTIUS99/21812 may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, 5 e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or 10 carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid 15 hydrophobic polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up 20 to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. The pharmaceutical compositions also may comprise 25 suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the 30 proteinase inhibiting compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Such pharmaceutically acceptable base -87- WO00/17357 PCT/US99/21812 addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium 5 hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like. The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present 10 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 15 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 20 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 immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T 25 cells can be combined with the pharmaceutical composition 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 pharmaceutically acceptable carriers, with 30 amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. -88- WO00/17357 PCT/US99/21812 Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal 5 formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. The amount of protein of the present invention in 10 the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of 15 the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered 20 until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 tg to 25 about 100 mg (preferably about 0.1 tg to about 10 mg, more preferably about 0.1 [tg to about 1 mg) of protein of the present invention per kg body weight. The therapeutic method includes administering the composition topically, systematically, or locally as an implant or 30 device, especially for compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration. When administered, the -89- WO00/17357 PCT/US99/21812 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 5 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 10 alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing 15 composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical 20 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 25 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 30 biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other -90- WO00/17357 PCTIUS99/21812 potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of 5 material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently 10 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 15 blood clot, to prevent the protein compositions from disassociating from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, 20 ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, 25 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 30 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 -91- WO00/17357 PCT/US99/21812 infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins of the invention may be combined with other agents 5 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 10 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 15 proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue 20 weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may 25 vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also 30 affect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for -92- WO00/17357 PCT/US99/21812 example, X-rays, histomorphometric determinations and tetracycline labeling. Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be 5 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 10 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. 15 5.6.3. EFFECTIVE DOSAGE Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to 20 achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the 25 capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be 30 formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test -93- WO00/17357 PCT/US99/21812 compound which achieves a half-maximal inhibition of the desired activity). Such information can be used to more accurately determine useful doses in humans. A therapeutically effective dose refers to that 5 amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for 10 determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 15 and ED 50 . Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range 20 of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the 25 individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to 30 maintain the therapeutic effects, or minimal effective concentration (MEC). The MEC will vary for each compound -94- WO00/17357 PCTIUS99/21812 but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma 5 concentrations. Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably 10 between 50-90%.In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. The amount of composition administered will, of course, be dependent on the subject being treated, on the 15 subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. 5.6.4. PACKAGING 20 The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may 25 be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. -95- WO00/17357 PCT/US99/21812 5.7. ANTIBODIES Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies include monoclonal and polyclonal 5 antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR and/or antigen-binding sequences, which 10 specifically recognize a polypeptide of the invention. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published June 20, 1993, which is incorporated herein by reference in its 15 entirety. Antibody fragments, including Fab, Fab , F(ab)2, and Fv, are also provided by the invention. The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind EGF-Hyl polypeptides exclusively (i.e., able to 20 distinguish an EGF-Hyl polypeptide from other EGF repeat containing polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in 25 ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely 30 practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold -96- WO00/17357 PCTIUS99/21812 Spring Harbor , NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the EGF-Hyl polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as 5 defined above, EGF-Hyl polypeptides. As with antibodies that are specific for full length EGF-repeat-containing polypeptides, antibodies of the invention that recognize EGF-Hyl fragments are those which can distinguish EGF-Hyl polypeptides from the family of EGF-repeat-containing 10 polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins. Antibodies of the invention can be produced using any method well known and routinely practiced in the art. Non-human antibodies may be humanized by any methods 15 known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. 20 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 25 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, 30 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 -97- WO00/17357 PCT/US99/21812 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 5 cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be 10 mediated by the protein. In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A.M., Monoclonal Antibodies Technology: Laboratory Techniques in 15 Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 20 Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96). Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with a peptide or 25 polypeptide of the invention. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the 30 present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection. The protein that is -98- WO00/17357 PCT/US99/21812 used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are 5 not limited to, coupling the antigen with a heterologous protein (such as globulin or -galactosidase) or through the inclusion of an adjuvant during immunization. For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, 10 such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening 15 the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures 20 known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. 25 Patent 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention. For polyclonal antibodies, antibody containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired 30 specificity using one of the above-described procedures. The present invention further provides the above described antibodies in delectably labeled form. -99- WO00/17357 PCT/US99/21812 Antibodies can be delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as 5 FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Sternberger, L.A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E.A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., 10 Immunol. 109:129 (1972); Goding, J.W. J. Immunol. Meth. 13:215 (1976)). The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the 15 polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The present invention further provides the above described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as 20 polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D.M. et al., " Handbook of Experimental Immunology" 4th Ed., 25 Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity 30 purification of the proteins of the present invention. -100- WO 00/17357 PCT/US99/21812 5.8. COMPUTER READABLE SEQUENCES In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, " computer 5 readable media" refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical 10 storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium 15 having recorded thereon a nucleotide sequence of the present invention. As used herein, " recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on 20 computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention. A variety of data storage structures are available to a skilled artisan for creating a computer readable 25 medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store 30 the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text -101- WO00/17357 PCT/US99/21812 file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled 5 artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention. By providing the nucleotide sequence 10 of SEQ ID NO:1 or 10, or a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 or 10 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software 15 is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et 20 al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in 25 fermentation reactions and in the production of commercially useful metabolites. As used herein, " a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information 30 of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, -102- WO00/17357 PCT/US99/21812 output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer 5 based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, " data storage means" 10 refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention. 15 As used herein, " search means" refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify 20 fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems 25 of the present invention. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for 30 conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a " target sequence" can be any nucleic acid or amino acid -103- WO00/17357 PCT/US99/21812 sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the 5 database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene 10 expression and protein processing, may be of shorter length. As used herein, " a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the 15 sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. 20 Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences). 5.9. TRIPLE HELIX FORMATION 25 In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. 30 Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in -104- WO00/17357 PCT/US99/21812 transcription (triple helix - see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Olmno, J. Neurochem. 56:560 5 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix- formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into 10 polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide. 15 5.10. DIAGNOSTIC ASSAYS AND KITS The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test 20 sample, using a nucleic acid probe or antibodies of the present invention. In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the 25 polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization 30 conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a -105- WO00/17357 PCTIUS99/21812 polynucleotide is amplified, a polynucleotide of the invention is detected in the sample. In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a 5 compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample. 10 In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample. 15 Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in 20 the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in 25 Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); 30 Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The -106- WO00/17357 PCTIUS99/21812 Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above 5 described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted 10 in order to obtain a sample which is compatible with the system utilized. In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. 15 Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more 20 of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody. In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic 25 containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a 30 quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the -107- WO00/17357 PCT/US99/21812 antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of 5 detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will 10 readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art. 15 5.11. SCREENING ASSAYS Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a protein comprising a polypeptide encoded by the ORF 20 from a nucleic acid with a sequence of SEQ ID NO:1 or 10, to an EGF repeat region or a specific domain of the polypeptide encoded by the nucleic acid, or to a nucleic acid comprising a sequence of SEQ ID NO:l or 10. In detail, said method comprises the steps of: 25 (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid. 30 In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide -108- WO00/17357 PCT/US99/21812 of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of 5 the invention is identified. Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to 10 form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified. Methods for identifying compounds that bind to a 15 polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the 20 complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified. Compounds identified via such methods can include 25 compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the 30 expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). -109- WO00/17357 PCT/US99/21812 Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression. 5 The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. 10 For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally 15 selected or designed. As used herein, an agent is said to be " rationally selected or designed" when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate 20 peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A 25 User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like. In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used 30 to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally -110- WO00/17357 PCT/US99/21812 designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF 5 for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of 10 sulfhydryl or polymeric derivatives which have base attachment capacity. Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in 15 transcription (triple helix - see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of 20 Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix- formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to 25 be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents. Agents which bind to a protein encoded by one of the ORFs of the 30 present invention can be used as a diagnostic agent, in the control of bacterial infection by modulating the activity of the protein encoded by the ORF. Agents which -111- WO00/17357 PCT/US99/21812 bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition. 5 5.12. USE OF NUCLEIC ACIDS AS PROBES Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The 10 hybridization probes of the subject invention may be derived from the nucleotide sequence of the SEQ ID NO:1 or 10. Because the corresponding gene is only expressed in a limited number of tissues, especially adult tissues, a hybridization probe derived from SEQ ID NO:1 or 10 can 15 be used as an indicator of the presence of RNA of cell type of such a tissue in a sample. Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described US Patent Nos 4,683,195 and 4,965,188 20 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both. The probe will comprise a discrete nucleotide sequence for the detection of identical 25 sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences. Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA 30 probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the -112- WO00/17357 PCT/US99/21812 appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic 5 sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal 10 markers, hybridization screening with libraries or flow sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) 15 Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map 20 data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA 25 associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. The nucleotide sequence may be used to produce purified polypeptides using well known methods 30 of recombinant DNA technology. Among the many publications that teach methods for the expression of genes after they have been isolated is Goeddel (1990) -113- WO00/17357 PCTIUS99/21812 Gene Expression Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego. polypeptides may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species 5 from which a particular polypeptide nucleotide sequence was isolated or from a different species. Advantages of producing polypeptides by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified 10 purification procedures. Each sequence so obtained was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT 670 Sequence Analysis System. In this algorithm, Pattern 15 Specification Language (developed by TRW Inc., Los Angeles, CA) was used to determine regions of homology. The three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance. Using a combination of these three 20 parameters, the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish 25 regions of homology from chance matches. Smith-Waterman alignments were used to display the results of the homology search. Peptide and protein sequence homologies were ascertained using the INHERIT- 670 Sequence Analysis System in a way similar to that used in DNA sequence 30 homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology that were scored -114- WO00/17357 PCT/US99/21812 with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches. Alternatively, BLAST, which stands for Basic Local 5 Alignment Search Tool, is used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol 215:403 10). BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. 10 Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. Whereas it is ideal for matches which do not contain gaps, it is inappropriate for performing motif-style searching. The fundamental unit 15 of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The 20 BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes 25 the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match 30 satisfies E is reported in the program output. In addition, BLAST analysis was used to search for related molecules within the libraries of the LIFESEQ -115- WO00/17357 PCT/US99/21812 database. This process, an " electronic northern" analysis is analogous to northern blot analysis in that it uses one cellubrevin sequence at a time to search for identical or homologous molecules at a set stringency. 5 The stringency of the electronic northern is based on " product score" . The product score is defined as (% nucleotide or amino acid [between the query and reference sequences] in Blast multiplied by the % maximum possible BLAST score [based on the lengths of query and reference 10 sequences]) divided by 100. At a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Homologous or related molecules can be identified by selecting those which show product scores between approximately 15 and 30. 15 The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is 20 intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. 6. EXAMPLE: A NOVEL POLYNUCLEOTIDE ENCODING A 25 POLYPEPTIDE CONTAINING AN EGF-REPEAT OBTAINED FROM A CDNA LIBRARY OF FETAL LIVER SPLEEN A plurality of novel nucleic acids were obtained 30 from the cDNA library prepared from human fetal liver spleen (20 week post-conception), as described in Bonaldo et al., Genome Res. 6:791-806 (1996), using standard PCR, SBH sequence signature analysis, and Sanger sequencing techniques. The inserts of the library were amplified -116- WO00/17357 PCT/US99/21812 with PCR using primers specific for vector sequences flanking the inserts. These samples were spotted onto nylon membranes and interrogated with oligonucleotide probes to give sequence signatures. The clones were 5 clustered into groups of similar or identical sequences, and single representative clones were selected from each group for gel sequencing. The 5' sequence of the amplified inserts was then deduced using the reverse M13 sequencing primer in a 10 typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single-pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer. Among these inserts identified as novel sequence not previously 15 obtained from this library, and not previously reported in public databases was the insert of clone pEGF-HY1. This clone was deposited with the ATCC as ATCC accession number 203198. Sequencing further with gene-specific primers (5' 20 ATGAGTGTAGCTCCAGTCCTTG-3' (SEQ ID NO:3) and 5' CACCATCAACACAGATTCCTT-3' (SEQ ID NO:4) yielded the nucleotide sequence depicted in Figure 1 as SEQ ID NO:1. The polypeptide encoded by SEQ ID NO:1 nucleotides 3-284 are is also shown in Figure 1 as SEQ ID NO:2. The 25 amino acid sequence bears similarity with the epidermal growth factor-repeat of the Notch protein, a protein involved in cell-fate control mechanism regulating multicellular development. -117- WO 00/17357 PCT/US99/21812 7. EXAMPLE: EXPRESSION OF THE NOVEL GENE COMPRISING SEQ ID NO:1 To study the role of SEQ ID NO:1 and the polypeptide 5 it expresses, the expression of the gene comprising SEQ ID NO:1 was analyzed using a semi-quantitative polymerase chain reaction-based technique. Human cDNA libraries were used as sources of expressed genes from tissues of interest (adult brain, adult heart, adult kidney, adult 10 lymph node, adult liver, adult lung, adult ovary, adult placenta, adult spleen, adult testis, bone marrow, fetal kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage). Gene specific primers were used to amplify a 420 nt portion of 15 the SEQ ID NO:1 sequence (5'-CCCAAACCCCTCAGTGTG-3', SEQ ID NO:5 and 5'-GTCTCTGTCCACTTG-3', SEQ ID NO:6) from the samples. Amplified products were separated on an agarose gel. SEQ ID NO:1 was expressed in a limited set of human 20 tissues. Of the 18 human tissues tested, fetal liver, fetal spleen fetal lung, fetal skin, fetal brain and adult lung provided a PCR amplified product, indicating that expression of SEQ ID NO:1 is not ubiquitous and is possibly limited to mostly developmental tissues. 25 Further testing of a variety of cancerous tissues is also carried out. 8. EXAMPLE: THE POLYPEPTIDE COMPRISING SEQ ID NO:2 CONTAINS AMINO ACID SEQUENCES 30 WITH HOMOLOGY TO THE EPIDERMAL GROWTH FACTOR (EGF)-REPEATS. The nucleotide sequence shown in Figure 1, and labeled SEQ ID NO:1, encodes the translated amino acid 35 sequence SEQ ID NO:2, which is also shown in Figure 1. -118- WO00/17357 PCT/US99/21812 In particular, SEQ ID NO:1 from nucleotides 3 to 284 encodes the 94 amino acid sequence depicted in SEQ ID NO:2. Sequence analysis of the amino acid sequence of SEQ ID NO:2 revealed that the sequence contains three 5 EGF-repeats. In particular, Figure 2 presents an amino acid alignment of SEQ ID NO:2 with the EGF-repeats of Notch, CD97 and EGF. The alignment reveals a high degree of homology between the repeats. The most striking feature 10 of this homology is its cysteine and glycine amino acid residue conservation among the repeats (underlined in Figure 2). 9. EXAMPLE: ADDITIONAL SEQUENCE OF EGF-Hyl 15 Nucleotides 1-193 of SEQ ID NO:10 were obtained using RACE. The nucleotides 193-1120 of SEQ ID NO: 10 were obtained in separate RACE reactions in three successive steps. Nucleotides 882-1120 were obtained using Fetal Brain Marathon-Ready cDNA (CLONETECH, Palo 20 Alto, CA) as template in RACE with adaptor primer AP2 (CLONETECH) and a gene-specific primer (5' GTAAAGACCTAGTTGGGGAATTC-3'; SEQ ID NO: 12). The resultant PCR products were cloned and sequenced. Nucleotides 674-881 were similarly obtained with adaptor 25 primer AP2 and a gene-specific primer (5' CAGCGGCAGAGGAAAGTGTGGTG-3'; SEQ ID NO: 13). Nucleotides 193-673 were similarly obtained with adaptor primer AP2 and a gene-specific primer (5'-GAGCTGCCTCTCGTGTCCAGA-3'; SEQ ID NO:14). 30 The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and -119- WO00/17357 PCT/US99/21812 compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those 5 skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims. All references cited within the body of the instant 10 specification are hereby incorporated by reference in their entirety. -120-

Claims (18)

1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide selected 5 from the group consisting of: (1) polypeptides comprising the amino acid sequence of SEQ ID NO:2, (2) polypeptides comprising amino acid residues 1-14 of SEQ ID NO:2, 10 (3) polypeptides comprising amino acid residues 15 54 of SEQ ID NO:2, (4) polypeptides comprising amino acid residues 55 94 of SEQ ID NO:2, (5) polypeptides comprising the amino acid sequence 15 encoded by the cDNA insert of clone pEGF-HY1 deposited under ATCC accession no. 203198, (6) polypeptides comprising the amino acid sequence of SEQ ID NO:11, (7) polypeptides comprising amino acid residues 349 20 385 of SEQ ID NO:11, (8) polypeptides comprising amino acid residues 387 423 of SEQ ID NO:11, and (9) polypeptides comprising amino acid residues 425 467 of SEQ ID NO:11; or 25 (b) a nucleotide sequence selected from the group consisting of: (1) the nucleotide sequence of SEQ ID NO:1, (2) nucleotides 3-284 of SEQ ID NO:1, (3) nucleotides 3-44 of SEQ ID NO:1, 30 (4) nucleotides 45-164 of SEQ ID NO:l, (5) nucleotides 165-284 of SEQ ID NO:1, (6) the protein coding sequence of the cDNA insert of clone pEGF-HY1 deposited under ATCC accession no. 203198, 35 (7) the nucleotide sequence of SEQ ID NO:10, (8) nucleotides 4-1407 of SEQ ID NO:10, (9) nucleotides 1048-1158 of SEQ ID NO:10, (10) nucleotides 1162-1270 of SEQ ID NO:10, and (11) nucleotides 1276-1404 of SEQ ID NO:10. 40

2. An isolated polynucleotide which hybridizes to the complement of the polynucleotide of Claim 1 under stringent hybridization conditions and which encodes a polypeptide with EGF-Hyl activity. -121- WO 00/17357 PCT/US99/21812

3. An isolated polynucleotide which comprises the complement of the polynucleotide of Claim 1. 5

4. A vector comprising the isolated polynucleotide of Claim 1 or 2.

5. An expression vector comprising the isolated polynucleotide of Claim 1 or 2. 10

6. A host cell genetically engineered to contain the polynucleotide of Claim 1 or 2.

7. A host cell genetically engineered to contain 15 the polynucleotide of Claim 1 or 2 in operative association with a regulatory sequence that controls expression of the polynucleotide in the host cell.

8. An isolated polypeptide comprising: 20 (1) the amino acid sequence of SEQ ID NO:2, (2) amino acid residues 1-14 of SEQ ID NO:2, (3) amino acid residues 15-54 of SEQ ID NO:2, (4) amino acid residues 55-94 of SEQ ID NO:2, (5) the amino acid sequence encoded by the cDNA 25 insert of clone pEGF-HY1 deposited under ATCC accession no. 203198, (6) the amino acid sequence of SEQ ID NO:11, (7) amino acid residues 349-385 of SEQ ID NO:11, 30 (8) amino acid residues 387-423 of SEQ ID NO:11, or (9) amino acid residues 425-467 of SEQ ID NO:11. 35

9. A composition comprising the polypeptide of Claim 8 and a carrier. -122- WO 00/17357 PCT/US99/21812

10. An antibody directed against the polypeptide of Claim 8.

11. A method for detecting a polynucleotide of 5 Claim 1 or 2 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex; and 10 b) detecting the complex, so that if a complex is detected, a polynucleotide of Claim 1 or 2 is detected.

12. A method for detecting a polynucleotide of 15 Claim 1 or 2 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of Claim 1 or 2 under such conditions; and 20 b) amplifying the annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of Claim 1 or 2 is detected. 25

13. The method of Claim 12, wherein the polynucleotide is an RNA molecule that encodes a polypeptide of Claim 8, and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide. -123- WO 00/17357 PCT/US99/21812

14. A method for detecting a polypeptide of Claim 8 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the 5 polypeptide for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, a polypeptide of Claim 8 is detected. 10

15. A method for identifying a compound that binds to a polypeptide of Claim 8, comprising: a) contacting a compound with a polypeptide of Claim 8 for a time sufficient to form a 15 polypeptide/compound complex; and b) detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polypeptide of Claim 8 is identified. 20

16. A method for identifying a compound that binds to a polypeptide of Claim 8, comprising: a) contacting a compound with a polypeptide of Claim 8, in a cell, for a time 25 sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting 30 reporter gene sequence expression, so that if a polypeptide/compound complex is -124- WO00/17357 PCT/US99/21812 detected, a compound that binds to a polypeptide of Claim 8 is identified.

17. A method of modulating activity of a 5 polypeptide of Claim 8, comprising contacting a cell that expresses the polypeptide with a compound that modulates activity of the polypeptide for a time sufficient to modulate said activity. 10

18. A method of modulating activity of the polypeptide of Claim 8, comprising contacting the polypeptide with a compound that modulates activity of the polypeptide for a time sufficient to modulate said activity. -125-