NZ503984A - Viral encoded semaphorin protein receptor (VESPR) DNA and polypeptides to treat inflammatory conditions - Google Patents

Abstract

Described are VESPR polypeptides as a purified and isolated protein, the DNA encoding the VESPR polypeptide, host cells transfected with cDNAs encoding VESPR, and methods for preparing VESPR polypeptides.

Description

New Zealand Paient Spedficaiion for Paient Number 503984 WO 99/21997 PCT/US98/22879 VIRAL ENCODED SEMAPHORIN PROTEIN RECEPTOR DNA AND POLYPEPTIDES FIELD OF THE INVENTION The present invention relates to semaphorin receptor polypeptides, the nucleic acids encoding such semaphorin receptor polypeptides, processes for producing recombinant semaphorin receptor polypeptides, and pharmaceutical compositions containing such polypeptides.

BACKGROUND OF THE INVENTION The semaphorin gene family includes a large number of molecules that encode related transmembrane and secreted glycoproteins known to be neurologic regulators The semaphorins are generally well conserved in their extracellular domains which 20 are typically about 500 amino acids in length Semaphorin family proteins have been observed in neuronal and nonneuronal tissue and have been studied largely for their role in neuronal growth cone guidance For example, the secreted semaphorins known as collapsin-1 and Drosophila semaphonn II are selectively involved in repulsive growth cone guidance dunng development. Flies having semaphonn II 25 genes that are mutated so that their function is reduced exhibit abnormal behavior characteristics Another semaphonn gene has been identified in several strains of poxvirus This semaphonn is found in vaccinia virus (Copenhagen strain) and is encoded m an open reading frame (ORF) known as A39R The A39R encoded protein has no 30 transmembrane domain and no potential membrane linkage and is known to be a secreted protein A vanola virus ORF also contains sequences that share homology with the vaccinia virus ORF A39R at the nucleotide level and the amino acid level. Another viral semaphonn, AHV-sema, has been found in the Alcclaphine Herpesvirus (AHV) Genes encoding mammalian (human, rat, and mouse) semaphonns have been identified, based upon their similanty to insect semaphonns Functional studies of these semaphonns suggest that embryonic and adult neurons require a semaphonn to establish workable connections Significantly, the fast response time of growth cone cultures to appropnate semaphonns suggests that semaphonn signaling involves a 40 receptor-mediated signal transduction mechanism To date, one semaphonn receptor, designated neuropihn, has been isolated using mRNA from rat spinal cord Another Printed from Mimosa receptor, designated neuropihn-2, has been suggested (KoJodkin et al. Cell 90.153-762,1997) Semaphonn ligands that are secreted into the extracellular milieu signal through receptor beanng cells in a local and systemic fashion In order to further investigate the nature of cellular processes regulated by such local and systemic 0 signaling, it would be beneficial to identify additional semaphonn receptors and ligands Furthermore, because virus encoded semaphonns are produced by infected cells and are present in viruses that are lytic (poxviruses) and viruses that are not known to be neurotropic (AHV), it is unlikely that their pnmary function is to modify neurologic responses It is more likely that the virus encoded semaphonns function to 5 modify the immunologic lesponse of the infected host and it is likely that mammalian homologues to virus encoded semaphonns function to modify the immunologic response In view of the suggestion that viral semaphonns may function in the immune system as natuial immunoregulators it would be beneficial to identify semaphonn receptors as therapeutic agents for enhancing or downregulating the 0 immune response SUMMARY OF THE INVENTION The present invention pertains to semaphonn receptors as isolated or homogeneous proteins In particular, the present invention provides a semaphonn 5 receptor polypeptide, designated VESPR (Viral Encoded Semaphonn Protein Receptor) that binds semaphonns, including, but not limited to, the A39R vaccinia semaphonn and AHV semaphonn Also, within the scope of the present invention are DNAs encoding VESPR polypeptides and expression vectois that include DNA encoding VESPR polypeptides. The present invention also includes host cells that 0 have been transfected oi transformed with expression vectors that include DNA encoding a VESPR polypeptide, and processes for producing VESPR polypeptides by cultunng such host cells under conditions conducive to expression The present invention further includes antibodies directed against VESPR polypeptides.

Further within the scope of the present invention are processes for punfying or 5 separating semaphonns or cells that express semaphonns to which the VESPR polypeptides of the present invention bind Such processes include binding at least one VESPR polypeptide to a solid phase matnx and contacting a mixture containing a semaphonn polypeptide to which the VESPR polypeptide binds, or a mixture of cells expressing the semaphonn with the bound VESPR polypeptide, and then separating 0 the contacting surface and the solution 2 Printed from Mimosa WO 99/21997 PCT/US98/22879 The present invention additionally provides processes for treating inflammation and inflammatory diseases. Such processes include administering a therapeutically effective amount of a soluble VESPR polypeptide to an human or other mammal afflicted with a disease associated with proinflammatory activity of a semaphonn ligand DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel semaphonn receptor polypeptides designated Viral Encoded Semaphonn Protein Receptor (VESPR), DNA encoding 15 VESPR polypeptides and lecombmant expression vectors that include DNA encoding VESPR polypeptides The present invention additionally provides methods for isolating VESPR polypeptides and methods for producing recombinant VESPR polypeptides by cultivating host cells transfected with the recombinant expression vectors under conditions appropnate for expressing semaphonn receptors and 20 recovenng the expressed receptor polypeptide.

In particular, the present invention provides VESPR polypeptides that bind semaphonns, including but not limited to, the vaccinia virus A39R semaphonn and the AHV semaphonn The native VESPR polypeptide described herein was isolated using an Ectromelia virus A39R semaphonn/Fc fusion protein (A39R/Fc) to recover 25 VESPR from the membranes of human cells expressing the receptor As descnbed m the examples below, flow cytometry expenments establish that the VESPR polypeptide polypeptides of the present invention are expressed by B cells lines, monocyte-type cell lines, T cell lines, dendntic cells NK cells, lung epithelial cells, stroma, intestinal epithelial cells and lymphoma cells 30 Furthermore, as demonstrated in the examples below, VESPR polypeptides of the present invention bind with their ligands to participate in upregulating the CD69 activation antigen on dendntic cells Also charactenstic of semaphonn receptors descnbed herein is their ability to interact with their ligands to synergize with interferon and SAC to upregulate IL-12 production and down regulate MHC class II 35 and CD86 expression on mouse dendntic cells VESPR polypeptides of the present invention are also associated with increased expression of CD54 on monocytes which suggests cellular activation as a result of the interaction between semaphonns and their receptors Among the uses of the VESPR polypeptides that flow from aforementioned biological properties of the receptor-ligand interaction are inducing 40 IL-12 production and subsequent natural killer cell activation. VESPR polypeptides find further use in treating diseases and adverse conditions associated with 3 Printed from Mimosa 2631-WO inflammation. In particular, soluble VESPR polypeptides can be used to antagonize proinflammatory activities associated with the interaction of semaphorin ligands and their receptors. Rheumatoid arthritis, a disease associated with chronic inflammation of synovial tissue, has been linked with upregulation of the human semaphorin E gene (Mangasser-Stephan et ah, Biochem and Bicphys Res Com, 234:153-156, 1997). Thus, soluble forms of VESPR polypeptides of the present invention may be useful in downregulating semaphorin activity that mediates this inflammatory disease.

VESPR, a native semaphorin receptor of the present invention, was isolated using a viral semaphorin ligand known as Ectromelia A39R. Example 1 below describes isolating the A39R semaphorin ligand and preparing an A39R/Fc fusion protein which was used to identify cell lines that bind the ligand and to determine the effects of interactions between A39R and its cell bound receptor.

Examples 4 and 5 describe identifying a native VESPR polypeptide of the present invention and isolating and purifying a human VESPR polypeptide. The amino acid sequence of the human VESPR polypeptide, isolated as descnbed in Example 5, is disclosed in SEQ ID NO:2. The amino acid sequence of SEQ ID NO:2 was obtained by sequencing the isolated and purified receptor using tandem mass spectrometry analysis of peptides produced in a trypsin digestion, in combination with contiguous EST sequences and identified cDNAs. The amino acid sequence presented in SEQ ID NO:2 has a predicted extracellular domain of amino acids 1-944 that includes a signal peptide with a cleavage site predicted at amino acid 34. The predicted transmembrane domain of SEQ ID NO:2 includes amino acids 945-965 and the cytoplasmic domain of SEQ ID NO:2 extends from ammo acids 966-1568.

A DNA encoding amino acids 19-1100 of human VESPR in E. coli DH10B was deposited with the American Type Culture Collection, Rockville, MD, USA on October 22, 1997, and assigned accession number 98560. The deposit was made under the terms of the Budapest Treaty The DNA construct of the deposit differs from that of SEQ ID NO:l in that nucleotide 172 is C. The resulting encoded amino acid 58 is leu.

Amino acid sequence searches were performed in available data bases for proteins and polypeptides sharing homology with the full length VESPR or domains thereof. The searches for polypeptides sharing homology with VESPR were performed using the BLAST algorithm described by Altschul et al., J Mol Bio 215: 403-410 (1990). This program was used to compare the VESPR amino acid sequence with protein a-nd DNA 4 a*-' «-•«_> • P H 2631-WO sequences found in data bases obtained from the National Center for Biotechnology Information. Similarity scores obtained as a result 4a ;WO 99/21997 of these searches identified groups of polypeptides having varying degrees of homology with VESPR The highest degree of similarity was found to be between the VESPR and a group of proteins known as the "plexin gene family" (Maestnni et al., 1996, and Kameyama et al., 1996) Pairwise and multiple sequence alignments between VESPR and human and murine members of the plexm family were 0 performed using the Smith-Waterman algorithm as implemented in the Genetics Computer Group (GCG) programs "BESTFIT" and "PELEUP" (Wisconsin Package, 9 0) The GCG program "DISTANCES" was used to calculate average pairwise percentage identity of the aligned protein sequences.

Pairwise sequence alignments between the VESPR polypeptide and each of 5 several members of the plexin gene family revealed an average identity in their cytoplasmic domain (amino acids 966-1568) of 39% to 40% and an average identity for each of the entire protein of 24% - 25% The higher degree of homology in the cytoplasmic domains suggests similar signal transduction mechanisms among the cytoplasmic domains 0 In order to identify regions of similarity between the protein sequences found to have some overall homology, homology analyses of the results of protein data base searches were performed using the BLIXEM and MSPCRUNCH programs (Sonnhammer and Durbin (1944a,b) The homology analyses revealed a novel subdomam with similarity to a region of the semaphonn domain of a number of 5 members of the semaphonn family of genes descnbed by Kolodkin et al. (1993) The novel subdomain includes amino acids 380-482 of the VESPR sequence of the present invention This subdomain can be subdivided into two distinct smaller regions, that include residues 388-402 and 454-482, respectively The C-proximal half-subdomain contains several highly conserved cysteine and tryptophan residues, 0 forming a consensus sequence of C-x(5)-C-x(2)-C-x(7)-C-x-W-C-x(5)-C, where x is any amino acid This entire subdomain is distinct from the canonical semaphonn domain descnbed for the semaphonn gene family in that (a) it is smaller (100 ammo acid residues for the subdomain vs 500 residues for the entire semaphonn domain), (b) it is also present in the plexin gene family and MET-hepatocyte growth factor 5 receptor family, neither of which is a canonical semaphonn gene family members, and (c) it is present m a gene which is not itself a member of the semaphonn gene family but which interacts with a member of the semaphonn family (A39R). These subdomain sequences, therefore, represent peptides that are potentially capable of further identifying other leceptors which interact with semaphonns.

Printed from Mimosa :WO 99/21997 A cDNA sequence that encodes the VESPR polypeptide of SEQ ID NO'2 was assembled as a composite of contiguous EST and cloned cDNA nucleotide sequences and is disclosed m SEQ ID NO 1. As descnbed m Example 5, identifying the cDNA that encodes the amino acid sequence of SEQ ED NO 2 enables constructing expression vectors that include the encoding cDNAs Then cultunng host cells 0 transfected with a recombinant expression vector that contains cDNA encoding VESPR polypeptide, under conditions appropnate for expressing the VESPR polypeptide, and recovenng the expressed VESPR polypeptide provides methods for producing VESPR polypeptides of the present invention Since VESPR polypeptide is found m B cell lines, T cell lines and dendntic 5 cells, treating a vanety of conditions associated with overactive or underactive immuno-regulation is possible. Moreover, the ligand and receptor complex may be involved in neural growth, development and/or maintenance While not limited to such, particular uses of the VESPR are descnbed infra The terms "VESPR" and "VESPR polypeptide" of the present invention 0 encompass polypeptides having the amino acid sequence SEQ ID NO'2, and proteins that are encoded by nucleic acids that contain the nucleic acid sequence of SEQ ID NO 1 In addition, the terms include those polypeptides that have a high degree of similanty or a high degree of identity with the ammo acid sequence of SEQ ID NO.2, which polypeptides are biologically active and bind at least one molecule or 5 fragments of a molecule that are members of the semaphonn family. In addition, the term VESPR refers to biologically active gene products of the DNA of SEQ ID NO:l Further encompassed by the term VESPR are soluble or truncated proteins that compnse pnmanly the binding portion of the protein, retain biological activity and are capable of being secreted Specific examples of such soluble proteins are those 0 compnsing the sequence of ammo acids 1-944 of SEQ ID NO.2 The term "biologically active" as it refers to VESPR or semaphonn receptor polypeptide, means that the VESPR or semaphonn receptor polypeptide is capable of binding to at least one semaphonn. Assays suitable for determining VESPR binding are descnbed herein and can include standard flow cytometry tests and slide binding 5 tests "Isolated" means a VESPR is substantially free of association with other proteins or polypeptides residual of the expression process, for example, as a punfication product of recombinant host cell culture or as a punfied extract.

A VESPR vanant as referred to herein, means a polypeptide substantially 0 homologous to native VESPR, but which has an amino acid sequence different from 6 Printed from Mimosa : WO 99/21997 PCT/US98/22879 that of native VESPR because of one or more deletions, insertions or substitutions. The vanant amino acid sequence preferably is at least 80% identical to a native VESPR amino acid sequence, most preferably at least 90% identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 8.1 descnbed by Devereux et al (Nucl Acids 10 Res 12 387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG) The preferred default parameters for the GAP program include (1) a unary companson matnx (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted companson matnx of Gnbskov and Burgess, Nucl Acids Res. 74.6745, 1986, as descnbed by Schwartz and Dayhoff, 15 eds , Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979, (2) a penalty of 3.0 for each gap and an additional 0 10 penalty for each symbol in each gap, and (3) no penalty for end gaps Vanants may compnse conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical charactenstics 20 Examples of conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity charactenstics, are well known Naturally occurnng 25 VESPR vanants or alleles are also encompassed by the invention Examples of such vanants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the VESPR protein, wherein the binding property is retained Alternate splicing of mRNA may yield a truncated but biologically active VESPR polypeptide, such as a naturally occurnng soluble form of the protein, for example 30 Vanations attnbutable to proteolysis include, for example, differences in the N-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the VESPR polypeptide (generally from 1-5 terminal amino acids).

As mentioned above, Example 1 descnbes the construction of novel viral 35 A39R/Fc fusion proteins useful m studying VESPR binding. Other antibody Fc regions may be substituted for the human IgGl Fc region descnbed in the Example Suitable Fc regions are those that can bind with high affinity to protein A or protein G, and include the Fc region of human IgGl or fragments of the human or munne IgGl Fc region, e g , fragments compnsing at least the hinge region so that interchain 40 disulfide bonds will form The viral A39R.Fc fusion protein offers the advantage of 7 Printed from Mimosa being easily purified. In addition, disulfide bonds form between the Fc regions of two separate fusion protein chains, creating dimers As descnbed above, in one aspect, the present invention includes soluble VESPR polypeptides. Soluble VESPR polypeptides compnse all or part of the extracellular domain of a native VESPR but lack the transmembrane region that 10 would cause retention of the polypeptide on a cell membrane. Soluble VESPR polypeptides advantageously compnse the native (or a heterologous) signal peptide when initially synthesized to promote secretion, but the signal peptide is cleaved upon secretion of VESPR polypeptides from the cell. Soluble VESPR polypeptides encompassed by the invention retain the ability to bind semaphonn ligands. Indeed, 15 soluble VESPR polypeptides may also include part of the signal or part of the cytoplasmic domain or other sequences, provided that the soluble VESPR protein can be secreted Soluble VESPR may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired 20 protein from the culture medium, e g , by centnfugation, and assaying the medium (supernatant) for the presence of the desired protein The presence of VESPR in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein Soluble forms of VESPR polypeptides possess many advantages over the 25 native, membrane bound VESPR protein Punfication of the proteins from recombinant host cells is feasible, since the soluble proteins are secreted from the cells Further, soluble proteins arc generally more suitable for intravenous administration Examples of soluble VESPR polypeptides include those compnsing a 30 substantial portion of the extracellular domain of a native VESPR polypeptide. An example of a soluble VESPR polypeptide is amino acids 1-944 of SEQ ID NO 2. In addition, truncated soluble VESPR proteins compnsing less than the entire extracellular domain are included in the invention, e.g. ammo acids 35-944 When initially expressed within a host cell, soluble VESPR polypeptides may additionally 35 compnse one of the heterologous signal peptides descnbed below that is functional within the host cells employed Alternatively, the protein may compnse the native signal peptide In one embodiment of the invention, soluble VESPR can be expressed as a fusion protein compnsing (from N- to C-terminus) the yeast a-factor signal peptide, a FLAG® peptide descnbed below and in U S Patent No 5,011,912, and 40 soluble VESPR polypeptide consisting of amino acids 1-944 or 35-944 of SEQ ID Printed from Mimosa NO:2 This recombinant fusion protein is expressed in and secreted from yeast cells The FLAG® peptide facilitates purification of the protein, and subsequently may be cleaved from the soluble VESPR using bovine mucosal enterokmase Isolated DNA sequences encoding soluble VESPR proteins are encompassed by the invention Truncated VESPR polypeptides, including soluble polypeptides, may be 0 prepared by any of a number of conventional techniques A desired DNA sequence may be chemically synthesized using techniques known per se DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA 5 fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein The well known polymerase chain reaction procedure also may be employed to amplify a DNA sequence encoding a desired protein fragment As a further alternative, known mutagenesis techniques may be employed to insert a stop codon at a desired point, e.g., immediately downstream of the codon for the last 0 amino acid of the binding domain As stated above, the invention provides isolated or homogeneous VESPR polypeptides, both recombinant and non-recombmant Vanants and derivatives of native VESPR proteins that retain the desired biological activity (e g., the ability to bind to semaphonns) may be obtained by mutations of nucleotide sequences coding 5 for native VESPR polypeptides. Alterations of the native amino acid sequence may be accomplished by any of a number of conventional methods Mutations can be introduced at particulai loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restnction sites enabling ligation to fragments of the native sequence Following ligation, the resulting reconstructed sequence encodes an analog 0 having the desired amino acid insertion, substitution, or deletion Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42.133, 1986); Bauer 5 et al (Gene 37'73, 1985), Craik (BioTechniques, January 1985, 12-19), Smith et al (Genetic Engineering- Principles and Methods, Plenum Press, 1981), Kunkel (Proc. Natl Acad Sci USA 82 488, 1985), Kunkel et al (Methods in Enzymol. 154361, 1987), and U S Patent Nos. 4,518,584 and 4,737,462 all of which are incorporated by reference 9 Printed from Mimosa WO 99/21997 PCT/US98/22879 Native VESPR polypeptide may be modified to create VESPR denvatives by forming covalent or aggiegative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent denvatives of VESPR polypeptides may be prepared by linking the chemical moieties to functional groups on VESPR amino acid side chains or at the N-terminus or C-terminus of a 10 VESPR polypeptide or the extracellular domain thereof Other denvatives of VESPR polypeptides within the scope of this invention include covalent or aggregative conjugates of VESPR polypeptides or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-termmal or C-terminal fusions For example, the conjugate may comprise a signal or leader polypeptide 15 sequence (e g. the a-factor leader of Saccharomyces) at the N-terminus of a VESPR polypeptide The signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall VESPR polypeptide fusions can compnse peptides added to facilitate 20 punfication and identification of VESPR Such peptides include, for example, poly-His or the antigenic identification peptides descnbed m U S Patent No 5,011,912 and m Hopp et al., Bio/Technology 6 1204, 1988 The invention further includes VESPR with or without associated native-pattern glycosylation VESPR polypeptide expressed in yeast or mammalian 25 expression systems (e.g , COS-7 cells) may be similar to or significantly different from a native VESPR polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system Expression of VESPR polypeptides in bactenal expression systems, such as E. coli, provides non-glycosylated molecules Equivalent DNA constructs that encode vanous additions or substitutions of 30 amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity or binding are encompassed by the invention For example, N-glycosylation sites in the VESPR extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog m mammalian and yeast expression systems. N-glycosylation sites in 35 eukaryotic polypeptides are charactenzed by an amino acid tnplet Asn-X-Y, wherein X is any ammo acid except Pro and Y is Ser or Thr The native human VESPR protein compnses 24 such tnplets, at amino acids 86-88, 141-143,149-151, 241-243, 252-254, 386-388, 407-409, 548-550, 553-555, 582-584, 588-590, 591-593, 653,655, 686-688, 692-694, 715-717, 741-743, 771-773, 796-798, 821-823, 871-873, 890-892, 40 895-897 and 920-922 of SEQ ID NO 2 Appropnate substitutions, additions or Printed from Mimosa . WO 99/21997 PCT/US98/22879 deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site Known procedures for inactivating N-glycosylation sites in proteins include those descnbed in U S Patent 5,071,972 and 10 EP 276,846, hereby incorporated by reference In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bndges upon renaturation Other equivalents are prepared by modification of 15 adjacent dibasic amino acid residues to enhance expression in yeast systems m which KEX2 protease activity is present EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these 20 adjacent basic residues Lys-Lys painngs are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites The human VESPR contains 11 KEX2 protease processing sites Nucleic acid sequences within the scope of the invention include isolated 25 DNA and RNA sequences that hybndize to the VESPR nucleotide sequences disclosed herein under conditions of moderate or high stnngency, and that encode biologically active VESPR. Conditions of moderate stnngency, as defined by Sambrook et al Molecular Cloning A Laboratory Manual, 2 ed Vol. 1, pp 101 -104, Cold Spnng Harbor Laboratory Press, (1989), include use of a prewashing 30 solution of 5 X SSC, 0 5% SDS, 1 0 mM EDTA (pH 8 0) and hybndization conditions of about 55°C, 5 X SSC, overnight Conditions of high stnngency include higher temperatures of hybndization and washing. The skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the nucleic acid molecule and the 35 relative amount of A, T/U, C and G nucleotides.

Due to the known degeneracy of the genetic code wherein more than one codon can encode the same amino acid, a DNA sequence may vary from that shown in SEQ ID NO 1 and still encode a VESPR polypeptide having the amino acid sequence of SEQ ID NO 2 Such vanant DNA sequences may result from silent 11 Printed from Mimosa WO 99/21997 PCT/US98/22879 mutations (e g , occurnng dunng PCR amplification), or may be the product of deliberate mutagenesis of a native sequence The invention provides equivalent isolated DNA sequences encoding biologically active VESPR, selected from (a) cDNA compnsing the nucleotide sequence presented in SEQ ID NO 1, (b) DNA capable of hybndization to a DNA of 10 (a) under moderately stnngent conditions and that encodes biologically active VESPR polypeptide, (c) DNA that is degenerate as a result of the genetic code to a DNA defined in (a) or (b) and that encodes biologically active VESPR polypeptide, and (d) DNA complementary to the DNA of (a), (b) or (c) VESPR polypeptides encoded by such DNA equivalent sequences are encompassed by the invention. 15 DNAs that are equivalents to the DNA sequence of SEQ ID NO 1 will hybndize under moderately and highly stnngent conditions to the DNA sequence that encodes polypeptides comprising the sequence of SEQ ID NO.2 Examples of VESPR proteins encoded by such DNA, include, but are not limited to, VESPR fragments and VESPR proteins compnsing inactivated N-glycosylation site(s), 20 inactivated KEX2 protease processing site(s), or conservative amino acid substitution(s), as descnbed above VESPR polypeptides encoded by DNA denved from other species, wherein the DNA will hybndize to the cDNA of SEQ ID NO'l are also encompassed Variants possessing the requisite ability to bind semaphonns may be identified 25 by any suitable assay Biological activity of VESPR polypeptides may be determined, for example, by competition for binding to the receptor binding domain of semaphonns (i e. competitive binding assays) One type of a competitive binding assay for a VESPR polypeptide uses a radiolabeled, soluble VESPR and intact semaphonn-expressing cells Instead of 30 intact cells, one could substitute soluble semaphonn Fc fusion proteins bound to a solid phase through the interaction of a Protein A, Protein G or an antibody to the semaphonn or Fc portions of the molecule, with the Fc region of the fusion protein. Another type of competitive binding assay utilizes radiolabeled soluble semaphonns such as a fusion protein, and intact cells expressing VESPR 35 Competitive binding assays can be performed following conventional methodology. In one embodiment, a soluble VESPR polypeptide can be made to compete with an immobilized receptor for binding with a soluble semaphonn ligand For example, a radiolabeled soluble semaphonn ligand can be antagonized by soluble VESPR m an assay for binding activity against a surface-bound semaphonn receptor. 12 Printed from Mimosa Qualitative results can be obtained by competitive autoradiographic plate binding assays, or Scatchard plots may be utilized to generate quantitative results.

Alternatively, semaphonn binding proteins, such as VESPR or anti-semaphonn antibodies, can be bound to a solid phase such as a column chromatography matnx or a similar substrate suitable for identifying, separating or 0 punfying cells that express semaphonn on their surface Binding of a semaphonn-binding protein to a solid phase contacting surface can be accomplished by any means, for example, by constructing a VESPR :Fc fusion protein and binding such to the solid phase through the interaction of Protein A or Protein G Vanous other means for fixing proteins to a solid phase are well known in the art and are suitable 5 for use m the present invention For example, magnetic microspheres can be coated with VESPR and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures containing semaphonn-expressing cells are contacted with the solid phase that has VESPR polypeptides thereon Cells having semaphonn on their surface bind to the fixed VESPR and unbound cells then are washed away This 0 affinity-binding method is useful for punfying, screening or separating such semaphonn-expressing cells from solution Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes Such enzymes are preferably non-toxic and non-mjunous to the cells and are preferably directed to cleaving the cell-surface binding partner In the case of 5 semaphonn-VESPR interactions, the enzyme preferably would cleave the semaphonn, thereby freeing the resulting cell suspension from the "foreign" semaphonn receptor matenal The punfied cell population then may be used to repopulate mature (adult) tissues Alternatively, mixtures of cells suspected of containing semaphonn-positive 0 cells first can be incubated with biotinylated VESPR. Incubation penods are typically at least one hour m duration to ensure sufficient binding to semaphonn The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidm provides the binding of the cell to the beads. Use of avidin-coated beads is known in the art See Berenson, et al. J. Cell. Biochem, 5 10D 239 (1986) Washing unbound matenal and releasing the bound cells is performed using conventional methods As descnbed above, VESPR can be used to separate cells expressing semaphonn. In an alternative method, VESPR or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as to detect 0 semaphonn-expressing cells Radiolabeling with can be performed by any of 13 Printed from Mimosa WO 99/21997 PCT/US98/22879 several standard methodologies that yield a functional ^5VESPR molecule labeled to high specific activity Or an lodinated or biotinylated antibody against the semaphonn receptor can be used Another detectable moiety such as an enzyme that can catalyze a colonmetnc or fluorometnc reaction, biotin or avidin may be used Cells to be tested for semaphonn-expression can be contacted with labeled VESPR 10 polypeptide After incubation, unbound labeled VESPR is removed and binding is measured using the detectable moiety.

The binding charactenstics of VESPR (including vanants) may also be determined using a conjugated semaphorin (for example, 125j.semaphonn pc) in competition assays similar to those descnbed above In this case, however, intact 15 cells expressing semaphonns bound to a sohd substrate are used to measure the extent to which a sample containing a putative VESPR vanant competes for binding with a conjugated semaphonn Other means of assaying for VESPR include the use of anti-VESPR antibodies, cell lines that proliferate in response to VESPR, or recombinant cell lines 20 that express semaphonn and proliferate m the presence of VESPR The VESPR proteins disclosed herein also may be employed to measure the biological activity of semaphonn proteins in terms of their binding affinity for VESPR. As one example, VESPR polypeptides of the present invention may be used in determining whether biological activity is retained after modification of a 25 semaphonn protein (e g , chemical modification, truncation, mutation, etc ) The biological activity of a semaphonn protein thus can be ascertained before it is used in a research study, or in the clinic, for example VESPR polypeptides of the present invention find use as reagents that may be employed by those conducting "quality assurance" studies, e g , to monitor shelf life 30 and stability of semaphorin protein under different conditions To illustrate, VESPR polypeptides may be employed m a binding affinity study to measure the biological activity of an semaphonn protein that has been stored at different temperatures, or produced in different cell types. The binding affinity of the modified semaphonn protein for VESPR is compared to that of an unmodified semaphonn protein to detect 35 any adverse impact of the modifications on biological activity of the semaphonn VESPR polypeptides also find use as earners for delivenng agents attached thereto to cells expressing semaphonns As descnbed in example 7 below, a putative human semaphonn is expressed in cells found in the placenta, testis, ovary and spleen VESPR polypeptides can thus can be used to deliver diagnostic or therapeutic 14 Printed from Mimosa -WO 99/21997 PCT/US98/22879 agents to these cells (or to other cell types found to express a semaphonn on a cell surfaces) in in vitro or in vivo procedures Diagnostic and therapeutic agents that may be attached to a VESPR polypeptide include, but are not limited to, drugs, toxins, radionuclides, chromophores, enzymes that catalyze a colonmetnc or fluorometnc reaction, and the 10 like, with the particular agent being chosen according to the intended application Examples of drugs include those used in treating various forms of cancer, e.g, nitrogen mustards such as L-phenylalamne nitrogen mustard or cyclophosphamide, intercalating agents such as cis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine, and antibiotics such as bleomycin, 15 doxorubicin, daunorubicin, and denvatives thereof Among the toxins are ncin, abnn, dipthena toxin, Pseudomonas aeruginosa exotoxin A, nbosomal inactivating proteins, mycotoxins such as tnchothecenes, and denvatives and fragments (e.g, single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 131^ 99mfCj lllin) and 76gr Radionuclides suitable for therapeutic use include, but are not limited to, 131^ 211^, 77gr> 186rC) 188rC) 212pb, 212Bi> 109Pd) 64CUj and 67Cu Such agents may be attached to the semaphonn receptor by any suitable conventional procedure VESPR, being a protein, compnses functional groups on amino acid side chains that can be reacted with functional groups on a desired agent 25 to form covalent bonds, for example Alternatively, the protein or agent may be denvatized to generate or attach a desired reactive functional group The denvatization may involve attachment of one of the bifunctional coupling reagents available for attaching vanous molecules to proteins (Pierce Chemical Company, Rockford, Illinois) A number of techniques for radiolabeling proteins are known 30 Radionuclide metals may be attached to the receptor by using a suitable bifunctional chelating agent, for example Conjugates compnsing VESPR and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropnate for the particular application 35 Another use of the VESPR of the present invention is as a research tool for studying the role that the receptor, in conjunction with semaphonns, may play in immune regulation and viral infection The VESPR polypeptides of the present invention also may be employed in in vitro assays for detection of semaphonn to which it binds or VESPR, or the interactions thereof Printed from Mimosa As descnbed in Example 16 semaphonns interact with their membrane bound receptors of the present invention to synergize with interferon and Staphylococcus aureus (type C) (SAC) in the production of IL-12 from dendntic cells The use of VESPR and its semaphonn ligand to induce IL-12 production promotes natural killer cell and T cell production and induces cytokine production (pnmanly y-interferon) 0 IL-12 and IL-12 induced y interferon production favors Thl cell differentiation, and downregulates the production of cytokines associated with Th2 cell differentiation IL-12 is known to act as both a proinflammatory cytokine and an immunomodulator Thus, a soluble VESPR can be used to antagonize IL-12 production and downregulate an organism's Thl cell differentiation Similarly, a soluble VESPR can be used to 5 promote production of cytokines associated with Th2 cell differentiation, thus discouraging proinflammatory activity. Also, VESPR m combination with its semaphonn ligand can be used to boost IL-12 production in combination with vaccination for those pathogens against which cellular immunity are effective. In this manner the enhanced amount of IL-12 acts as an adjuvant in the vaccination to induce 0 a more persistent Thl-type immunological memory Furthermore, it is known that administration of IL-12 to tumor beanng animals results in tumor regression and the establishment of a tumor-specific immune response. Thus, using a semaphonn ligand to bind with VESPR in order to enhance or promote IL-12 can induce a curative immune response against aggressive 5 micromctastasizing tumors Additionally, as descnbed m example 18, receptors of the present invention bind with their semaphonn ligands to increase CD54 expression on monocytes This observation suggests that the semaphonn/semaphonn receptor interaction mediate cellular activation that contnbutes to the proinflammatory activity typically associated 0 with monocyte activation Such activity includes increased phagocytosis, pmocytosis, nitnc oxide production and cytokine production To antagonize or reverse the proinflammatory activity resulting from the interaction between the semaphonn ligand and its membrane bound receptor, a pharmaceutical composition containing a therapeutically effect amount of a soluble VESPR of the present 5 invention can be administered parenterally to an organism The soluble VESPR binds with the semaphonn ligand thus preventing the ligand from binding with a membrane bound receptor and contnbuting to the proinflammatory activity. A therapeutically effect amount of VESPR is an amount sufficient to antagonize proinflammatory activity 16 Printed from Mimosa WO 99/21997 PCT/US98/22879 Notwithstanding the increased expression of CD54 on monocytes, microphysiometer data indicate that cellular signaling through VESPR does not activate the cell in a classical immunological sense More particularly, microphysiometer data suggests that when VESPR binds with its ligand a decrease in the rate of change of the pH of the medium results This is the opposite of that which 10 occurs dunng cell activation Such a decrease is experienced with drugs that inhibit protein kinases in the cell, or with viral infections which can paralyze the cell metabolically Suppressive signals that are delivered through a VESPR can be antagonized by the administration of soluble forms of VESPR Such soluble forms effectively bind the VESPR binding partner and antagonize the suppressive signaling, 15 thus preventing the inactivation status of the cell Alternatively, and in accordance with the present invention, anti-VESPR antibodies that signal can be used as a therapeutic to treat autoimmune diseases in which inflammation is a result of presentation of self antigens to T cells More particularly, such anti-VESPR antibodies can be targeted to be taken up by antigen presenting cells, and, like protein 20 kinase inhibitors, inactivate the antigen presenting cells The autoimmunity is cured because the antigen presenting cells responsible for the inflammation have become poor antigen presenters Semaphonn ligands binding with VESPR to downregulate expression of MHC Class II molecules and CD86, a co-stimulatory molecule, on dendntic cells, 25 cultured with GM-CSF and IL-4 (see example 17) suggests that the interaction between semaphonn ligands and the receptors of the present invention are associated with the immune suppression of mature dendntic cells To antagonize or reverse the immunosuppression activity resulting from the interaction between the semaphonn ligand and its membrane bound receptor, a pharmaceutical composition containing a 30 therapeutically effective amount of a soluble VESPR of the present invention can be administered parenterally to an organism The soluble VESPR binds with the semaphonn ligand thus preventing the ligand from binding with a membrane bound receptor and contnbuting to the immunosuppression activity. Alternatively, in patients or organisms that suffer from the effects of chronic inflammation, 35 administenng appropnate semaphonn ligands will contnbute to suppressing the proinflammatory activity of differentiated macrophages.

Data indicate that a VESPR ligand is found on T cells and VESPR is involved in the migration of dendntic cells from the T cell zones of lymph nodes Additionally, data suggests that VESPR is involved in shutting down the T cell 40 immune response once a pathogen has been cleared 17 Printed from Mimosa : WO 99/21997 PCT/US98/22879 VESPR polypeptides of the invention can be formulated according to known methods used to prepare pharmaceutically useful compositions VESPR can be combined m admixture, either as the sole active matenal or with other known active materials, with pharmaceutically suitable diluents (e g , Tns-HCl, acetate, phosphate), preservatives (e g , Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, 10 adjuvants and/or earners Suitable earners and their formulations are descnbed m Remington's Pharmaceutical Sciences, 16th ed 1980, Mack Publishing Co In addition, such compositions can contain VESPR polypeptide complexed with polyethylene glycol (PEG), metal ions, or incorporated mto polymenc compounds such as polyacetic acid, polyglycohc acid, hydrogels, etc, or incorporated into 15 liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts Such compositions will influence the physical state, solubility, stability, rale of in vivo release, and rate of in vivo clearance of VESPR VESPR polypeptide can also be conjugated to antibodies against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specific 20 receptors VESPR polypeptides can be administered topically, parenterally, or by inhalation The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, lntracisternal injection, or infusion techniques These compositions will typically contain an effective amount of the VESPR, alone or in combination 25 with an effective amount of any other active matenal Such dosages and desired drug concentrations contained in the compositions may vary depending upon many factors, including the intended use, patient's body weight and age, and route of administration Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art-accepted 30 practices.

VESPR polypeptides may exist as oligomers, such as covalently-linked or non-covalently-lmked dimers or tnmers Oligomers may be linked by disulfide bonds formed between cysteine residues on different VESPR molecules. In one embodiment of the invention, a VESPR dimer is created by fusing VESPR to the Fc 35 region of an antibody (e g , IgGl) m a manner that does not interfere with binding of VESPR to a semaphonn ligand-binding domain The Fc polypeptide preferably is fused to the C-terminus of a soluble VESPR (compnsing only the ligand-binding domain) General preparation of fusion proteins compnsing heterologous polypeptides fused to vanous portions of antibody-denved polypeptides (including 40 the Fc domain) has been descnbed, e g., by Ashkenazi et al. (PNAS USA 88:10535, 18 Printed from Mimosa WO 99/21997 PCT/US98/22879 1991) and Byrn et al (Nature 344:611, 1990), hereby incorporated by reference A gene fusion encoding the VESPR Fc fusion protein is inserted into an appropriate expression vector VESPR Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding divalent If fusion proteins are made with both heavy and 10 light chains of an antibody, it is possible to form a VESPR oligomer with as many as four VESPR extracellular regions Alternatively, one can link two soluble VESPR domains with a peptide linker Suitable host cells for expression of VESPR polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use 15 with bactenal, fungal, yeast, and mammalian cellular hosts are descnbed, for example, in Pouwels et al Cloning Vectors A Laboratory Manual, Elsevier, New York, (1985) Cell-free translation systems could also be employed to produce VESPR polypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, for example, 20 E coli or Bacillus Suitable prokaryotic host cells for transformation include, for example, E coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus In a prokaryotic host cell, such as E coli, a VESPR polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the 25 prokaryotic host cell The N-terminal methionine may be cleaved from the expressed recombinant VESPR polypeptide VESPR polypeptides may be expressed m yeast host cells, preferably from the Saccharomyces genus (e g„ S cerevisiae) Other genera of yeast, such as Pichia , K. lactis or Kluyveromyces, may also be employed Yeast vectors will often contain an 30 ongin of replication sequence from a 2\i yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionem, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255.2073, 1980) or other 35 glycolytic enzymes (Hess et al, J Adv Enzyme Reg. 7 149, 1968; and Holland et al, Biochem. 17 4900, 1978), such as enolasc, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucoses-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tnosephosphate isomerase, phosphoglucose isomerase, and glucokinase Other suitable vectors and 40 promoters for use in yeast expression are further descnbed in Hitzeman, EPA-73,657 19 Printed from Mimosa or m Fleei et. al, Gene, 107:285-195 (1991), and van den Berg et al., Bio/Technology, 8 135-139 (1990). Another alternative is the glucose-repressible ADH2 promoter descnbed by Russell et al. {J. Biol. Chem. 258.2614, 1982) and Beier et al (Nature 300 724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and 10 replication in E. coli (Ampr gene and ongin of replication) into the above-descnbed yeast vectors The yeast a-factor leader sequence may be employed to direct secretion of the VESPR polypeptide The a-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g , Kurjan et al., Cell 15 30-933, 1982, Bitter et al , Proc Natl Acad. Sci USA 81'5330, 1984, U. S Patent 4,546,082, and EP 324,274 Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art A leader sequence may be modified near its 3' end to contain one or more restnction sites This will facilitate fusion of the leader sequence to the structural gene. 20 Yeast transformation protocols are known to those of skill in the art One such protocol is descnbed by Hinnen et al., Proc Natl Acad Sci USA 75.1929, 1978 The Hinnen et al protocol selects for Trp+ transformants in a selective medium, wherein the selective medium consists of 0 67% yeast nitrogen base, 0 5% casamino acids, 2% glucose, 10 ng/ml adenine and 20 (ig/ml uracil 25 Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a "nch" medium An example of a nch medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ug/ml adenine and 80 ug/ml uracil Depression of the ADH2 promoter occurs when glucose is exhausted from the medium 30 Mammalian or insect host cell culture systems could also be employed to express recombinant VESPR polypeptides Baculovirus systems for production of heterologous proteins m insect cells are reviewed by Luckow and Summers, Bio/Technology 6A1 (1988). Established cell lines of mammalian ongin also may be employed Examples of suitable mammalian host cell lines include the COS-7 line of 35 monkey kidney cells (ATCC CRL 1651) (Gluzman et al, Cell 23:115, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-l/EBNA-1 cell line denved from the Afncan green monkey kidney cell line CVI (ATCC CCL 70) as descnbed by McMahan et al. (EMBO J 10. 2821, 1991) Printed from Mimosa WO 99/21997 PCT/US98/22879 Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are denved from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus DNA sequences denved from the SV40 viral genome, for example, SV40 ongin, early and late promoter, enhancer, 10 splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral ongin of replication (Fiers et al, Nature 273 113, 1978) Smaller or larger SV40 fragments may also be used, 15 provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral ongin of replication site is included.

Exemplary expression vectors for use m mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol Cell Biol. 5.280, 1983) A useful system for stable high level expression of mammalian cDNAs in CI27 munne 20 mammary epithelial cells can be constructed substantially as descnbed by Cosman et al. (Mol Immunol 23 935, 1986) A useful high expression vector, PMLSV N1/N4, descnbed by Cosman et al, Nature 572-768, 1984 has been deposited as ATCC 39890 Additional useful mammalian expression vectors are descnbed in EP-A-0367566, and in U S. Patent Application Senal No 07/701,415, filed May 16, 1991, 25 incorporated by reference herein The vectors may be denved from retroviruses In place of the native signal sequence, and in addition to an initiator methionine, a heterologous signal sequence may be added, such as the signal sequence for IL-7 descnbed in United States Patent 4,965,195, the signal sequence for IL-2 receptor descnbed in Cosman et al, Nature 312-768 (1984), the IL-4 signal peptide descnbed 30 in EP 367,566, the type I IL-1 receptor signal peptide descnbed in U S Patent 4,968,607, and the type II IL-1 receptor signal peptide descnbed in EP 460,846 VESPR polypeptides as isolated, punfied or homogeneous proteins according to the invention may be produced by recombinant expression systems as descnbed above or punfied from naturally occurnng cells VESPR can be punfied to 35 substantial homogeneity, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) One process for producing VESPR compnses cultunng a host cell transformed with an expression vector compnsing a DNA sequence that encodes VESPR polypeptide under conditions sufficient to promote expression of VESPR 40 polypeptide The receptor is then recovered from culture medium or cell extracts, 21 Printed from Mimosa WO 99/21997 PCT/US98/22879 depending upon the expression system employed As is known to the skilled artisan, procedures for punfying a lccombmant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium.

For example, when expression systems that secrete the recombinant protein 10 are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Milhpore Pellicon ultrafiltration unit Following the concentration step, the concentrate can be applied to a punfication matnx such as a gel filtration medium Alternatively, an anion exchange resin can be employed, for example, a matnx or substrate having pendant 15 diethylaminoethyl (DEAE) groups The matnces can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein punfication Alternatively, a cation exchange step can be employed Suitable cation exchangers include vanous insoluble matrices compnsing sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred Finally, one or more reversed-phase high 20 performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e g., silica gel having pendant methyl or other aliphatic groups) can be employed to further punfy VESPR polypeptide Some or all of the foregoing punfication steps, in vanous combinations, are well known and can be employed to provide a substantially homogeneous recombinant protein 25 It is possible to utilize an affinity column compnsing the receptor-binding domain of a semaphonn that binds VESPR to affinity-punfy expressed VESPR polypeptides VESPR polypeptides can be removed from an affinity column using conventional techniques, e g, in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the 30 affinity matnx utilized Alternatively, the affinity column may compnse an antibody that binds VESPR Example 20 descnbes a procedure for employing VESPR of the invention to generate monoclonal antibodies directed against VESPR Recombinant piotein produced in bactenal culture can be isolated by initial disruption of the host cells, centnfugation, extraction from cell pellets if an insoluble 35 polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity punfication or size exclusion chromatography steps Finally, RP-HPLC can be employed for final punfication steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. 22 Printed from Mimosa : WO 99/21997 PCT/US98/22879 Transformed yeast host cells are preferably employed to express VESPR as a secreted polypeptide in order to simplify punfication. Secreted recombinant polypeptide from a yeast host cell fermentation can be punfied by methods analogous to those disclosed by Urdal et al (/ Chromatog 296:111, 1984). Urdal et al descnbe two sequential, reversed-phase HPLC steps for punfication of recombinant 10 human IL-2 on a preparative HPLC column Useful fragments of the VESPR nucleic acids include antisense or sense oligonucleotides compnsing a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target VESPR mRNA (sense) or VESPR DNA (antisense) sequences Antisense or sense oligonucleotides, according to the present 15 invention, compnse a fiagment of the coding region of VESPR cDNA Such a fragment generally compnses at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides The ability to denve an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is descnbed in, for example, Stein and Cohen (Cancer Res 48.2659, 1988) and van der Krol et al (BioTechmqu.es 20 6 958,1988) Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcnption or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcnption or translation, or by other means The 25 antisense oligonucleotides thus may be used to block expression of VESPR proteins Antisense or sense oligonucleotides further compnse oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those descnbed in W091/06629) and wherein such sugar linkages are resistant to endogenous nucleases Such oligonucleotides with resistant sugar linkages are stable 30 in vivo (i e, capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those descnbed in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid 35 sequence, such as poly-(L-lysine) Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence Antisense or sense oligonucleotides may be introduced into a cell containing 40 the target nucleic acid sequence by any gene transfer method, including, for example, 23 Printed from Mimosa WO 99/21997 PCT/US98/22879 CaP04-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex 10 vivo Suitable retroviral vectors include, but are not limited to, those denved from the munne retrovirus M-MuLV, N2 (a retrovirus denved from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656) Sense or antisense oligonucleotides also may be introduced into a cell 15 containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as descnbed in WO 91/04753 Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand 20 binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as descnbed in WO 90/10448. The sense or antisense ohgonucleotide-25 lipid complex is preferably dissociated within the cell by an endogenous lipase.

In addition to the above, the following examples are provided to illustrate particular embodiments and not to limit the scope of the invention EXAMPLE 1 Preparing an Ectromelia Semaphorin/Fc Fusion Protein The following descnbes preparation of an Ectromelia Semaphonn A39R/immunoglobulin fusion protein (A39R/Fc) The process included prepanng a DNA construct that encodes the fusion protein, transfecting a cell line with the DNA construct, and harvesting supernatants from the transfected cells. The A39R/Fc 35 fusion protein was used as descnbed in Examples 3, 4, 5 and 6 to study VESPR binding charactenstics and isolate VESPR DNA encoding A39R semaphonn was isolated and amplified from genomic Ectromelia virus DNA using PCR techniques and synthesized oligonucleotide pnmers whose sequences were based on published A39R sequences in the Copenhagen strain 40 of Vaccinia Virus The Copenhagen strain A39R DNA sequence is descnbed in 24 Printed from Mimosa Goebel, S.J. et al Virology 179.247, 1990. The isolated Ectromelia A39R DNA is presented in SEQ ID NO 7 and the protein encoded by the DNA is presented in SEQ ID NO 8 The upstream oligonucleotide primer introduced an Spel site upstream of amino acid 15 of the A39R polypeptide A downstream oligonucleotide primer introduced a Notl site downstream of the termination codon of Ectromelia A39R, after amino acid 399 The primer sequences were as follows Upstream Spel primer TGTCACTAGT ATCGAATGGC ATA AOTTTGA A (SEQ ID NO.3) Spe 1 A39R DNA Downstream Notl primer GAGAGCGGCC GCCTATTACA TTTTA AGTAT TTT (SEQ ID NO 4) Not 1 A39R DNA A Bgl II to Nsl I restriction fragment containing a mutem human Fc region of immunoglobulin as descnbed by Baum et al Cir Sh. 44.30 (1994) was ligated into an expression vector (pDC304) containing a munne IL-7 signal peptide and a FLAG™ octapeptide as dcscnbed in U S Patent No 5,011,912 The PCR amplified DNA encoding amino acids 15-399 of Ectromelia A39R was then ligated into the expression vector containing the mutein human Fc region, the munne IL-7 signal peptide and FLAG™ peptide, m a two way ligation The resulting DNA construct was transfected into the monkey kidney cell lines CV-1/EBNA (with co-transfection of psv3neo) After 7 days of culture in medium containing 0.5% low immunoglobulin bovine serum, a solution of 0 2% azide was added to the supernatant and the supernatant was filtered through a 0 22 (im filtei Then approximately 1 L of culture supernatant was passed through a BioCad Protein A HPLC protein punfication system using a 4 6 x 100 mm Protein A column (POROS 20A from PerSeptive Biosystems) at 10 mL/min The Protein A column binds the Fc Portion of the fusion protein m the supernatant, immobilizing the fusion protein and allowing other components of the supernatant to pass through the column. The column was washed with 30 mL of PBS solution and bound fusion protein was eluted from the HPLC column with citnc acid adjusted to pH 3 0. Eluted punfied fusion protein was neutralized as it eluted using 1M HEPES solution at pH 7 4 Printed from Mimosa WO 99/21997 PCT/US98/22879 EXAMPLE 2 Preparing An Ectromelia Semaphorin/polvHis Fusion Protein The following describes preparation of an Ectromelia A39R/polyHis fusion protein (A39R/polyHis). The process included preparing a DNA construct that encodes the fusion protein, transfecting a cell line with the DNA construct, and 10 harvesting supematants from the transfected cells DNA encoding Ectromelia A39R (amino acids 1-399 of A39R ORF, SEQ ID NO 8) was isolated and amplified from genomic Ectromelia virus DNA using PCR techniques and synthesized oligonucleotide primers. The primers added a Not 1 site at the 5' end and the motif Gly-Ser-6xHIS at the 3' end for use in punfication 15 processes After the Gly-Ser-6xHIS motif the pnmers added an in-frame termination codon and a Bgl 2 site The PCR product was cut and cloned m pDC409 expression vector (McMahon et al , EMBO J 70:2821,1991) The resulting DNA construct was transiently transfected into the monkey cell line COS-1 (ATCC CRL-1650). Following a 7 day culture in medium containing 20 0 5% low immunoglobulin bovine serum, cell supematants were harvested and a solution of 0 2% sodium azide was added to the supematants. The supematants were filtered through a 0 22 |am filter, concentrated 10 fold with a prep scale concentrator (Millipore, Bedford, MA) and punfied on a BioCad HPLC protein punfication equipped with a Nickel NTA Superflow self pack resin column (Qiagen, Santa 25 Clanta, CA). After the supernatant passed through the column, the column was washed with Buffer A (20mM NaP04, pH7 4; 300mMNaCl, 50mM Imidazole) Bound protein was then eluted from the column using a gradient elution techniques Fractions containing protein were collected and analyzed on a 4-20% SDS-PAGE reducing gel Peaks containing A39R/polyHis fusion protein were pooled, 30 concentrated 2 fold, and then dialyzed in PBS The resulting A39R7polyHis fusion protein was then filtered through a 0 22|am stenle filter EXAMPLE 3 Screening Cell Lines for Binding to A39R The A39R/Fc fusion protein prepared as descnbed in Example 1 was used to screen cell lines for binding using quantitative binding studies according to standard flow cytometry methodologies For each cell line screened, the procedure involved incubating approximately 100,000 of the cells blocked with 2% FCS (fetal calf serum), 5% normal goat serum and 5% rabbit serum in PBS for 1 hour. Then the 40 blocked cells were incubated with 5 ug/mL of A39R/Fc fusion protein in 2% FCS, 26 Printed from Mimosa 5% goat serum and 5% rabbit serum in PBS Following the incubation the sample was washed 2 times with FACS buffer (2% FCS in PBS) and then treated with mouse anti human Fc/biotin (purchased from Jackson Research) and SAPE (streptavidin-phycoerythnn purchased from Molecular Probes) This treatment causes the antihuman Fc/biotm to bind to any bound A39R/Fc and the SAPE to bind to the anti-10 human Fc/biotin resulting in a fluorescent identifying label on A39R/Fc which is bound to cells The cells were analyzed for any bound protein using fluorescent detection flow cytometry The results indicated the A39R semaphonn binds well to human NK cells, murine splenic B cells, human PB T cells, human T, B, erythroid, lymphoid and myeloid precursor cells, fibroblasts and epithelial lineage Table I 15 details the results of the flow cytometry studies A "+" indicates that binding was detected between the cell surface and A39R. A indicates that no binding was detected between the cell surface and A39R TABLE I Cell Line A39R Binding Result Namalwa (B cell-like lymphoma - human) + CB23 (Human Cord Blood B Cell Line) + EU-1 (Human pre B Cell Line) + MP-1 (Human B Cell Lymphoma) + PB B (Human Penpheral Blood B Cells) + Mouse Splenic B Cells + Mouse Splenic B Cells + CD40L + U937 (Human Monocyte-Type Cell) + HSB2 (Human T Cell Line) + K299 (Non Hodgkin's Lymphoma) + TE71 (Mouse Thymic Epithelium) + IEC18 (Rat Intestinal Epithelium) + IMTLH (Human Bone Marrow Denved Stroma) + W126 (Human Lung Epithelium) + PL-1 (Human T-Cell Clone + VK-1 Human T-Cell Clone + Pnmary Penpheral Blood T Cells + Pnmary Human NK Cells + RAJI (Burkitt's Lymphoma) 40 KG1 (Human myeloid Cell Line) 27 Printed from Mimosa WO 99/21997 PCT/US98/22879 THP-1 (Human Promonocytic Cell Line) + MC6 (Mouse Mast Cell) EL4 (Mouse Thymoma) BeWo (Chono Carcinoma) Pnmary Mouse Dendntic Cells + Pnmary Human Dendntic Cells + EXAMPLE 4 Identifying A Putative Semaphorin Receptor CB23 cells (human cord blood B cell line) and human PB T cells that tested positive for binding to A39R were tested for expression of a putative receptor and to determine if any receptor is expressed as a membrane bound molecule, soluble molecule, or both Broadly, the analyses involved radiolabehng CB23 and human PB T cell surfaces, harvesting and treating cell supematants and lysates with an A39R/Fc fusion protein to precipitate any putative receptor, and then visualizing an immunoprecipitate on an electrophoretic gel In particular, the procedure involved first radiolabehng approximately lxlO7 CB23 or PB T cells with [125I] as descnbed by Benjamin et al; Blood 75,:2017-2023 (1990) Cultured cell supematants were harvested and clanfied by centnfugation at 14,000 rpm for 30 minutes Cell lysates were generated by incubating the cells on ice for 30 minutes in 1 mm L phosphate-buffered saline with 1% Tnton-X 100 containing protease inhibitors phenylmethylsulfonyl fluonde, Pepstatin-A, and Leupeptin. The lysates were clanfied by centnfugation at 14,000 rpm for 30 minutes In order to precipitate any receptor present m the lysate and/or supernatant, 200|aL of the cell supernatant or lysate was incubated with 2p.g of A39R/Fc fusion protein prepared as descnbed in Example 1 The incubation was carried out for 1 hour with gentle rocking at 4°C An Fc protein control sample was prepared and incubation in the same manner Following the incubation, Protein-A Sepharose beads (#17-0780-01 Pharmacia Biotech Inc., Piscataway, NJ) were added to the lysates and supematants and the mixture was incubated for 1 hour with gentle rocking at 4°C The beads were washed extensively with a PBS 1% Tnton-X 100 solution Bound protein was eluted and analyzed by SDS PAGE. Protein bands were visualized by autoradiography and a single, approximately 200K Da band was found to bind to A39R/Fc but not to the control Fc Protein. The semaphonn receptor was present in cell lysate and cell supernatant, confirming its expression as membrane bound protein and as a secreted 40 soluble protein 28 Printed from Mimosa -WO 99/21997 Example 5 Isolating and Sequencing a Semaphorin Receptor The A39R/Fc fusion protein, prepared as descnbed in Example 1, was used to isolate a human semaphonn receptor polypeptide and a procedure for the isolated polypeptide purification was confirmed. The semaphonn receptor was isolated by suspending CB23 cell pellets m a solution of protease inhibitors that included 1 mM each of PMSF, Leupeptin, Aprotinin, Pepstatin A, 10|ig/mL APMSF, and 1 mM EDTA in homogenization buffer (10 mM phosphate, 30 mM NaCl, pH 7.4). The cells were dounce homogenized and layered over a solution of 41% sucrose in homogenization buffer and the spun down in a Beckman SW-28 rotor at 25,000 rpm, at 4°C for 45 minutes The interphases were collected and diluted in cold homogenization buffer, dounced, and spun The resulting clean membrane pellets were stored at -80 °C Membrane pellets prepared from 240 mLs of packed cells were combined with 100 mLs of an aqueous solution of 20 mM Tns, 150 mM NaCl, the protease inhibitors identified above, 1% Triton X-100 and 0 1 mM of CaCl2, MgCl2, and McCl2 salts (Buffer A) The suspended pellets were dounced and spun m a SW-28 rotor for 30 minutes at 25,000 rpm at 4°C The supernatant was placed onto a lOOmL wheat germ agglutinin column and allowed to elute at a rate of 1 mL/mmute with 10 column volumes of Buffer A Pioteins that were specifically bound to the column were then eluted with Buffer A containing 0.2 M N-acetyl glucosamine Fractions testing positive for protein were pooled and incubated with 100 pg of A39R/Fc fusion protein for 1 hour at 4°C The incubated mixture was run through a sepharose column to remove matenal that did not specifically bind and then allowed to pass through a 0 5 mL column of Protein A/Sepharose solid support The Protein A/Sepharose solid support was washed with 20 column volumes of PBS containing 1% Tnton X-100 followed by a wash with PBS to wash off any unbound matenal. Then proteins that were retained on the Protein A/Sepharose column were eluted m a stepwise manner with 0.35 mL fractions of 50 mM citrate at pH 3.0. Fractions that tested positive for protein were combined and concentrated to 50 pL using a 10 kD MWCO Centncon concentrator Protein in the resulting concentrated sample were reduced and then alkylated using standard DTT and lodoacetic acid procedures The alkylation proteins were then electrophoresed on an 8% gel Proteins on the gel were visualized with coomassie-G in 50% MeOH containing 5% acetic acid and then destained in 50% MeOH 29 Printed from Mimosa - WO 99/21997 The approximately 200 kD band, located by companson to protein standards, was excised with a razor blade and washed overnight in 100 mM ammonium carbonate The gel slice was speed evaporated until dry and a 1 10 solution of trypsin in 100 mM ammonium carbonate was added to the dned slide. The slide was incubated at 37°C for 16 hours and then protein in the slice was extracted with 50% 0 acetonitnle with 5% formic acid three times while incubating 30 minutes with each extraction.

The trypsin digested peptide fragments were lyophilized, reconstituted in 50(iL of 0 1% tnfluoroacetic acid, and separated by RP-HPLC on a 500 ^ id x 25 cm capillary column packed with C-18 reverse phase packing. The HPLC liquid phase 5 was an acetonitnle/water gradient of 10% after 5 minutes, 85% after 105 minutes Eluting protein was detected at 215 nm Each protein was collected as it eluted in separate fractions and N-termmal sequence analysis of the peptides in the fraction was performed on a 494 Procise sequencer according to the manufacturer's instruction.

RP-HPLC fractions, obtained as descnbed above, were dned on a vacuum 0 centnfuge and peptides in the fraction were dissolved in 6|xL of 50% methanol containing 0 5% acetic acid Two microliters (2 (iL) of each of the peptide solutions were loaded into nanospray tips (Protein Analysis Company, Odense, Denmark) Data were obtained with a Finnigan TSQ700 tnple quadrupole mass spectrometer (San Jose, CA) equipped with a nanospray source Mass spectra were acquired at unit 5 resolution For tandem mass spectrometry, the first quadrupole was operated at a resolution sufficient to pass a 3-4 Da wide window, and the third quadrupole was operated at unit resolution Collision gas was supplied at a pressure of 4 mTorr Methyl estenfication was performed using standard estenfication procedures The tandem mass spectrometry analysis of the trypsin generated peptides 0 provided amino acid sequence information for isolated portions of the punfied protein The tandem mass spectral data were used in computer assisted screening of non-redundant protein databases and EST databases using the local SEQUEST algonthm search tool (Eng, J.K et al J am Soc. Mass 1994) The peptide query sequences GluGluThrProValPheTyrLys corresponding to amino acids 421-428 of 5 SEQ ID NO 2, and AsnlleTyrlleTyrLeuThrAlaGlyLys, corresponding to amino acids 436-445 identified EST No 248534 (Accession N78220) as containing peptide sequences having 100% identity to the query peptide sequences The peptide query sequence ThrValLeuPheLeuGlyThrGlyAspGlyGlnLeuLeuLys corresponding to ammo acids 388-401 identified EST No R08946 as containing a 100% identity to the 0 query Printed from Mimosa ;WO 99/21997 PCT/US98/22879 The 100% identity between portions of EST 248534 and three peptide fragments of the punfied protein strongly suggested that the cDNA contained within EST 248534 represented a portion of the nucleotide sequence for the coding region of the punfied protein A source of semaphonn receptor cDNA was identified using phage library screening methods and PCR pnmers based upon EST 248534 10 The oligonucleotide pnmers had the following nucleotide sequences.

ATCGCATCAT CTACCTTCAT CCATTCCGAC CTG (SEQ ID N0 9) TAAACACTCC GAACAGGATT TATGTTTATT GCA (SEQ ID NO 10) PCR isolation and amplification methodologies were earned out using a panel of human tissue cDNA phage libranes as templates for the PCR reactions The PCR reaction mixture included 1 j_lL of phage library stock, PCR oligonucleotide pnmers at 0 3 |_iM final concentration, lx PC2 buffer (Ab Peptides, Inc, St. Louis, MO), 0 2 20 mM each of dATP, dCTP, dGTP, dTTP (Pharmacia Biotech) 0.2 |iL of a 16T mix Klen-Taq/Vent polymerase (Klen-Taq polymerase, Ab Peptides, Inc. and Vent polymerase, New England Biolabs, Beverly, MA) in a 30 pL final reaction volume The PCR reaction cycles included one cycle at 98°C for 5 minutes; thirty cycles at 98°C for 45 seconds, thirty cycles at 68°C for 45 seconds; thirty cycles at 72°C for 45 25 seconds, and 1 cycle at 72°C for 5 minutes using a Robocycler 96 from Stratagene, La Jolla, CA cDNAs in several libranes were positively identified as containing DNA encoding the punfied VESPR protein based upon the appearance of an appropnate sized DNA band in electrophoresed PCR product.

Two of the phage libranes, human foreskin fibroblast and human dermal 30 fibroblasts, were chosen tor additional analysis Libranes were plated according to established prodedures and probed with a radiolabeled random pnmer probe denved from a PCR amplification product using EST 248534 as a template. The PCR conditions used to obtained the amplification product were as descnbed above and the probe was generated using Pnme-IT II Random Pnmer Labeling Kit from Stratagene, 35 La Jolla, CA Approximately 1 x 10° cpm/mL of punfied probe was used to probe human foreskin phage libranes on nylon membrane filters overnight at 63°C in a hybndization buffer of lOx Denhardts solution, 50 mM Tns at pH 7 5, 0.9 M NaCl, 0 01% Sodium Pyrophosphate, 1% sodium dodecyl sulfate, and 200|_ig/mL denatured, fragmented salmon sperm DNA After probing, the probed membranes were washed 40 once in 6x SSC, 0.1% SDS for 20 minutes, once m 2 x SSC, 0.1% SDS for 20 31 Printed from Mimosa :WO 99/21997 PCT/US98/22879 minutes, once in lx SSC, 0 1% SDS for 20 minutes, and once m 0.1 x SSC, 0.1% SDS for 20 minutes at 63°C The probed and washed filters were exposed to X-omat AR X-ray film (Eastman-Kodak Corp) overnight Four overlapping cDNAs were identified The overlapping cDNAs, together with the sequenced trypsin digest generated protein fragments were used to complete and confirm the coding sequence 10 of VESPR as shown in SEQ ID NO.l and the ammo acid sequence presented in SEQ ID NO 2 Example 6 Monoclonal Antibodies to A39R Semaphorin This example illustrates a method for preparing antibodies to A39R semaphonn Punfied A39R/Fc was prepared as descnbed m Example 1 above The punfied protein was used to generate antibodies against A39R semaphonn as descnbed in U S Patent 4,411,993 Bnefly, mice were immunized at 0, 2 and 6 weeks with 10 fig with A39R/Fc The pnmary immunization was prepared with 20 TITERMAX adjuvant, from Vaxcell, Inc , and subsequent immunizations were prepared with incomplete Freund's adjuvant (IFA) At 11 weeks, the mice were IV boosted with 3-4 \ig A39R/Fc in PBS Three days after the IV boost, splenocytes were harvested and fused with an Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution. Hybndoma supematants were screened for A39R 25 antibodies by dot blot assay against A39R/FC and an irrelevant Fc protein Example 7 Northern Blot Analyses for Tissue Expressing Semaphorin Receptor The following descnbes Northern Blot expenments earned out to identify 30 tissue and cell types that express VESPR polypeptides of the present invention. The results confirm the cell binding results obtained using flow cytometry analysis and the A39R/Fc fusion protein As descnbed in Example 5, EST data base searches resulted in the discovery of an EST that was believed to be a partial clone of the VESPR of SEQ ID NO 2 35 (EST 248534) A nboprobe template was generated using PCR techniques and oligonucleotide pnmers that were based on nucleotides 1-372 of EST 248534 The upstream and down stream pnmers that encompasses nucleotides 1-372 of EST 248534 had the following sequences 40 GCGGGACTCA GAGTCACC (SEQH>NO:5) 32 Printed from Mimosa :WO 99/21997 PCT/US98/22879 GGATCCTAAT ACGACTCACT ATAGGGAGGA AACCACTCCG AAC (SEQ ID NO-6) The underlined portion is a T7 site The two pnmers were used to isolate and amplify a PCR product from 10 EST248534 for use in generating a nboprobe The nboprobe was generated using Ambion's MAXIscnpt SP6/T7 kit by combining 3joL of RNAse free water, 2(iL lOx transcnption buffer, lpL each of lOmM dATP/dCTP/dGTP, 5pL 573' EST 248534 PCR Product, 5 |iL Amersham [a32P]UTP lOmCi/mL, 2|iL T7 RNA polymerase at room temperature The combination was microfuged, spun bnefly, and incubated at 15 37°C for 30 minutes Then IjiL DNAse was added to the mixture and allowed to react for 15 minutes at 37°C The reaction product was passed through two column volumes of G-25 packing (Boehnnger) One microliter (l|xL) of the nboprobe was counted in a scintillation counter for 1 minute to determine cpm/mL Northern blots were generated by fractionating polyadenylated RNA from a 20 vanety of cell lines on a 1 2% agarose formaldehyde gel and blotting the RNA onto Hybond Nylon membranes (Amersham, Arlington Heights, IL) Standard northern blot generating procedures as descnbed in Maniatis, (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Lab Press, 1989) were used. Total RNA multiple tissue northern blots were purchased commercially (BioChain Institute, Inc., 25 San Leandro, CA Cat #s 021001, 021002, 021003) The Northern blots were prehybndized in a 50% form amide hybndization solution (30 mL 20x SSC, 2 mL lOOx Denhardt's reagent, 1 mL of 10 mg/mL denatured fragmented salmon sperm DNA, 50 mL 100% formamide and 20 mL 10% SDS The total RNA blots were pre-hybndized at 42°C for 4 hours and the polyA+ 30 RNA blots were pre-hybndized at 63°C for 4 hours. The nboprobe was added to clean hybndization solution (same as prehybndization solution) at a count of 106 cpm/mL The prehybndization solution was removed from the blots and the hybndization solution and nboprobe were added to the blots The hybndization was allowed to proceed overnight with gentle shaking The total RNA blots hybridized at 35 63°C and the polyA+ RNA blots hybndized at 63°C The probed total RNA blots were washed once for 30 minutes in 2xSSC containing 0 05% SDS at 42°C and once foi 30 minutes in 2xSSC containing 0 05% SDS at 55°C, twice for 30 minutes m 0 IxSSC containing 0 1% SDS at 63°C, three times for 30 minutes m 0 IxSSC containing 0 1%SDS and then exposed to X-ray 40 film. The poly A+ blots were washed once for 30 minutes in a 2xSSC solution 33 Printed from Mimosa WO 99/21997 PCT/US98/22879 containing 0.05% SDS at 63°C and once for 30 minutes in IxSSC containing 0 1% SDS and then exposed to x-ray film The results of probing the Northern blots and visualizing the resulting x-ray film for positively binding probes confirm that VESPR is expressed in the same cells as those that showed positive binding in flow cytometry experiments Hybridizing 10 RNA was detected in MP-1, HFF and CB23 cells. Pnmary tissues showing positive RNA included heart, brain, lung, spleen and placenta No RNA was detecting in RAJ1 cells Example 8 Generating AHV Semaphorin Fc Fusion Protein The following describes preparing an AHV Semaphonn/immunoglobulin fusion protein (AHVSema/Fc) The process included preparing a DNA construct that encodes the fusion protein, transfecting a cell line with the DNA construct, and harvesting supematants from the transfected cells.

DNA encoding AHV-Sema is descnbed in Ensser et al J Gen Vir 76 1063- 1067, 1995 DNA encoding AHV-Sema amino acids 70-653 was isolated and amplified from Alcelaphine herpesvirus DNA strain WC11 (Plownght, W et al Nature 188 1167-1169, 1960) using PCR techniques and synthesized oligonucleotide pnmers whose sequences were based on the published AHV-Sema sequence. The 25 upstream oligonucleotide primer introduced a Spe 1 site A downstream oligonucleotide pnmer introduced a Not 1 site downstream of the termination codon The general method used to isolate the soluble AHVSema is descnbed in Spnggs et al, J Virology, 70 5557 (1996) A lestnction fragment containing a mutein human Fc region of 30 immunoglobulin as descnbed by Goodwin et al Cell 73, 447-456, 1993 was ligated into an expression vector (pDC409) containing a munne IL-7 signal peptide and a FLAG™ octapeptide as descnbed in U S Patent No 5,011,912. The PCR amplified AHVSema DNA encoding was then ligated into the expression vector containing the mutein human Fc region, the munne IL-7 signal peptide and FLAG™ peptide, m a 35 two way ligation. The resulting DNA construct was transfected into the monkey kidney cell lines CV-1/EBNA (with co-transfection of pSV3neo) After 7 days of culture in medium containing 0.5% low immunoglobulin bovine serum, a solution of 0 2% azide was added to the supernatant and the supernatant was filtered through a 0.22 p.m filter Then approximately 1 L of culture supernatant was passed through a 40 BioCad Protein A HPLC protein punfication system using a 4.6 x 100 mm Protein A 34 Printed from Mimosa column (POROS 20A from PerSeptive Biosystems) at 10 mL/min. The Protein A column binds the Fc Portion of the fusion protein in the supernatant, immobilizing the fusion protein and allowing other components of the supernatant to pass through the column The column was washed with 30 mL of PBS solution and bound fusion protein was eluted from the HPLC column with citric acid adjusted to pH 3.0 Eluted 0 purified fusions protein was neutralized as it eluted using 1M HEPES solution at pH 74 Example 9 Expressing Recombinant Semaphorin Receptor Using the semaphonn receptor (VESPR) ammo acid sequence of the protein punfied as descnbed in Example 5, and information denved from EST database searches and cDNAs obtained using hybridization methodologies with radiolabeled probes, also as descnbed m Example 5, cDNA is generated and cells are transfected with the cDNA to allow expression of recombinant VESPR polypeptide 0 The cDNA in DC409 expression vector, denved from pDC406, is transfected in CV1/EBNA cells using standard techniques (McMahan et al., EMBO J. 10 2821,1991) More particularly, CV1 EBNA cells are plated at a density of 2 x 106 cells per 10 cm dish in 10 mL of Dulbeccos Minimum Essential Medium (medium) supplemented with 10% fetal calf serum The cells are allowed to adhere overnight at 5 37°C The medium is replaced with 1.5 mL of medium containing 66 7 |iM chloroquine and a DNA mixture containing 5 |ig of cDNA encoding VESPR Medium containing 175|iL and 25 |iL of DEAE dextran is added to the cells The cells and cDNA are incubated at 37°C for 5 hours. The cDNA mixture is removed and cells are shocked with 1 mL of fresh medium containing 10% DMSO for 2 5 min 0 The medium is replaced with fresh medium and the cells are grown for at least 3 days To recover soluble forms of VESPR, supematants containing the soluble form are collected and the VESPR protein recovered using HPLC techniques or affinity chromatography techniques. To recover forms of VESPR that are membrane bound, 5 the transfected cells are harvested, fixed in 1 % paraformaldehyde, washed and used m their mtact form Example 10 VESPR Binding Studies In order to examine the binding charactenstics of a receptor polypeptide of the 0 present invention, binding studies were performed by subjecting cells expressing Printed from Mimosa membrane bound VESPR extracellular domain to the slide binding assay descnbed in Goodwin et al Cell 73 447-456, (1993) and Spnggs et al, J Virol 70:5557 (1996).

The pDC409 expression vector, denved from pDC406 (McMahon et al, EMBO J 10 2821, 1991) but having a single Bgl 2 was selected for the cloning process VESPR cDNA, encoding amino acids 19-1100, was subcloned into a 10 pDC409 expression vector through the Sal 1 (5') and Not 1 (3') sites, to form a DNA construct CV-1/EBNA cells were transfected via DEAE/Dextran with 2fig of a VESPR cDNA (encoding ammo acids 19-1100) in pDC409 (Gin et al., EMBO J 13 2822, 1994) The transfected cells were cultured for 3 days and the CV-1/EBNA cell 15 monolayers were incubated with 1 (ig/mL of A39R/Fc, AHVSema/Fc, or control Fc protein. Then the incubated cells were washed and incubated with 125I-labeled mouse anti-human IgG (Jackson Immunoresearch, West Grove, PA) After extensive washing, the cells were fixed, dipped in photographic emulsion as descnbed by Geanng et al, EMBO J 8 3667-3676 (1989) and developed Positive binding was 20 determined by the presence of exposed or darkened silver grains overlaying cells expressing VESPR that had bound Fc protein EXAMPLE 11 Flow Cytometry and Inhibition Binding Studies The following descnbes flow cytometnc analyses of CB23 cells for binding to A39R/Fc fusion protein (Example 1) and the AHVsema/Fc fusion protein (Example9) Also descnbed below is a study directed to determining inhibition of the AHVsema and A39R binding with and an excess of A39R/polyHis fusion protein prepared as descnbed m Example 2.

The flow cytometnc analysis was performed by first incubating about lxlO6 CB23 cells on ice for 30 minutes in FACS buffer and containing 3% normal goat serum and 3% normal rabbit serum to block non- specific binding Portions of A39R/Fc, AHVsema/Fc and a control Fc protein were added at varying concentrations and the incubation was continued for 30 minutes. The cells were 35 washed and then incubated with phycoerythnn-conjugated Fc specific anti-human IgG m FACS buffer The cells were washed and analyzed on a FACScan from Becton Dickinson, Bedford, MA The results showed positive binding of AHV semaphonn and the A39R semaphonn.

Binding inhibition studies were performed by incubating about lxlO6 CB23 40 cells for 30 minutes on ice in FACS buffer The A39R/polyHis and control HIS 36 Printed from Mimosa WO 99/21997 PCT/US98/22879 protein were added to different samples at varying concentrations and the incubation continued for another 30 minutes. Then A39R/Fc or AHVsema/Fc were added to the incubated cells at varying concentrations and the incubation was continued for another 30 minutes The cells were washed and then incubated with phycoerythnn-conjugated Fc specific anti-human IgG in FACS buffers The cells were washed 10 again and then analyzed on a FACScan The results demonstrated complete inhibition of A39R and AHVSema using A39R/polyHIS, but not the heterologous HIS containing protein Example 12 Human B Cell Aggregation with A39R Semaphorin In order to examine human B cell response to A39R semaphonn, human tonsillar B cells were punfied as descnbed m Spnggs et al., J Exp Med 176 1543, (1992) An A39R/polyHis fusion protein was prepared as descnbed in Example 2 A solution of A39R/polyHis fusion protein was prepared to a final A39R concentration 20 of 1 (ig/mL and the A39R/polyHis fusion protein solution was incubated in in vitro cultures of about 105 of the punfied B cells. Continuing the incubation for about 24 hours resulted in cellular aggregation When a 10 fold molar excess of the monoclonal antibody against A39R, prepared as descnbed in Example 6, was added to the fusion protein preparation pnor to adding the fusion protein to the cultures, the 25 cell aggregation was blocked Additionally, when the A39R semaphonn was heat inactivated pnor to adding it to the culture, the aggregation was blocked This work confirms that VESPR is expressed on B cells and that the interaction between A39R and VESPR results in B cell aggregation B cell aggregation is indicative of their activation Activated B cells are known to secrete 30 cytokines, produce antibodies, or become antigen presenting cells Example 13 Mouse Dendritic Cells and Macrophage Aggregation with A39R Semaphorin In order to examine dendntic cell and macrophage response to A39R, mouse cell cultures were brought into contact with A39R semaphonn and the effects of the combination noted Mouse dendntic cell cultures containing macrophages were obtained by immunizing mice with Flt3-L and cells were isolated and punfied as descnbed m Maraskovsky et al, J Exp Med 184 1953, (1996) 37 Printed from Mimosa WO 99/21997 PCT/US98/22879 Bnefly, female C57B1/6 mice were injected once daily with a solution of 10 |ig of Flt3L and 1 \ig mouse serum albumin m 100 (iL of PBS for 9-10 consecutive days After the immunization, single cell suspensions of spleens were prepared by disrupting spleen tissue between frosted glass slides in the presence of NH^Cl to deplete red blood cells The remaining cells were incubated with mAb to Thy-1, 10 B220, NK1 1, and TER119, and then incubated with 10% rabbit complement. Then the incubated cells were washed and residual mAb-coated cells were removed using anti-immunoglobulin (Ig)-coated magnetic beads The remaining ennched cells were cultured or sorted for the vanous cell populations.

Cells selected for sorting were stained with anti-CDllc and anti-CDllb and 15 sorted for the C and D/E populations as descnbed m Maraskovsky et al., J Exp Med 184.1953-1962, 1996 An A39R/polyHis lusion protein was prepared as descnbed in Example 2 An A39R/polyHis fusion protein solution was incubated in in vitro cultures at a final concentration of l^ig/mL with about 105 of the sorted or depleted mouse cells Within 20 4-6 hours the cells began to aggregate When a 10 fold molar excess of the monoclonal antibody against A39R, prepared as descnbed in Example 6, was added to the A39R/polyHis fusion protein preparation pnor to adding the fusion protein to the mouse cell cultures, the aggregation was blocked This work confirms that VESPR is expressed on dendntic cells and 25 macrophages, and that the interaction between A39R and VESPR results in dendntic cell and macrophage aggregation.

Example 14 A39R Semaphorin Upregulates CD69 Activation Antigen In order to investigate the effects of A39R semaphonn on cultured dendntic cells, mice were injected each day for 9 days with a Flt3-L preparation. Mouse dendntic cells were harvested and then cultured in medium containing 10% FBS and 20 ng/mL GM-CSF for 5 days.

On day 5, l(ig/niL of A39R/polyHis fusion protein was added to the culture 35 On day 6, the cells were stained with diagnostic antibodies The results of the diagnostic antibody staining expenments showed that CDllc\ CDllb+ cells (dendntic cells) expressed an increased amount of the CD69 activation antigen, thus demonstrating that the interaction of A39R semaphonn and its receptor upregulate CD69 expression 38 Printed from Mimosa :WO 99/21997 When the fusion protein is inactivated with heat, the fusion protein had no effect on the CD69 antigen Representative changes in mean fluorescence intensity between unstained and stained cells were from approximately 500 channels to 2500 channels Again, these results demonstrate significant effects of the interactions between A39R semaphonn and its membrane bound receptor on the regulation of the 10 CD69 activation antigen, a transient and early expressed marker for cell activation Example 15 Evaluating the Effect of A39R in the Production of IL-12 In order to study the role of A39R in the production of IL-12 from mouse 15 spleen cells, mice were immunized with flt3-L and dendntic cells were generated, harvested and punfied as descnbed in Example 13 Approximately 5xl05 cells/0 5 mL of punfied, unsorted dendntic cells were incubated in modified DMEM media (500 (iL at 1 x lOVmL) in the presence of one more of the following: 20ng/mL muGM-CSF (Immunex, Seattle, WA), 20ng/mL y -20 IFN (Genzyme, Boston, MA), lO^g/mL SAC (CalBiochem, La Jolla, CA) Each cell preparation was treated additionally with 1 [ig/mL of A39R/polyHis fusion protein alone or in combination with 1 (J-g/mL or 0.1 |ig/mL of muCD40L tnmer (Immunex, Seattle, WA) Cultures were incubated in humidified 37C, 10% C02-in-air for 16-18 h After incubation, the viability of each group of cultured cells was determined and 25 supematants were collected and assayed for muIL12 (P70) using an ELISA assay kit (Genzyme, Boston, MA) MuIL12 levels were calculated by reference to a standard curve constructed with recombinant cytokine ELISA testing demonstrated m particular that A39R interacts with its receptor to synergize with interferon and SAC in the production of IL-12 from unsorted mouse 30 dendntic cells This in vivo IL-12 induction promotes natural killer cell activation and gamma interferon production and contnbutes to upregulating gamma interferon sensitive cytokines Example 16 Testing Effects of A39R on Regulation of MHC Class II and CD86 on Monocytes The following expenment descnbes upregulation of MHC Class II and CD86 by the interaction of A39R with its membrane bound receptor Penpheral blood from healthy donors was diluted 1 1 in low endotoxin PBS at pH 7.4 and room 40 temperature Then 35 mLs of the diluted blood was layered over 15 mLs of Isolymph 39 Printed from Mimosa (Gallard and Schlesmger Industnes, Inc, Carle Place, NY) and centnfuged at 2200 rpm for 25 minutes at room temperature The plasma layers was reserved The PBMC layer was harvested and washed three times to remove the Isolymph The washed PBMC's were resuspended in X-Vivo 15 serum free media (BioWhittaker, Walkersviile, MD) and added to T175 flasks The flasks had been previously coated 10 with 2% Gelatin (Sigma, St. Louis, MO) and pre-treated for 30 minutes with the reserved plasma layer The PBMC's were allowed to adhere for 90 minutes at 37°C, 5% C02 and then nnsed three times gently with 10 mL washes of low endotoxin PBS Adhered monocytes were harvested by incubating the cells m Enzyme Free Dissociation Buffer (Gibco, BRL) and washing the cells multiple times m PBS 15 Monocytes were centnfuged at 2500 rpm for 5 minutes, counted, and set up in 24 well dishes at 5 x 10s cells/well in 1 mL The cultures were 95% pure Punfied monocytes were cultured for 7-9 days in the presence of 20 ng/mL GM-CSF and 100 ng/mL IL-4 in order to allow cells to differentiate to a more dendntic cell-likc phenotype On day 7-9, cultures were treated with 1 (jg/mL 20 A39R/polyHis or a control polyHis containing protein, and the next day cells and supematants were harvested for analyses In flow cytometnc expenments for examining monocyte-denved dendntic cell surface markers, cells were stained with conjugated mAbs directed against specific proteins The staining showed that foi a majonty of the penpheral blood donors 25 tested, A39R treatment downregulated CD86 and MHC class II expression on these cells. Since CD86 and MHC class II molecules are markers of an enhanced antigen presentation by dendntic cells, their downregulation suggests an immunosuppressive effect of the interaction of A39R with its receptor on this cell population Example 17 Unregulation of CD54 The following descnbes the effect of the interaction between A39R semaphonn and its receptor on punfied monocytes and more particularly, the impact of CD54 expression on monocytes after incubation with a semaphonn. Freshly 35 isolated monocytes were purified from penpheral blood donors as descnbed m Example 16, except that they were held in culture overnight in the presence of A39R/polyHis or control proteins Following the overnight culture, flow cytometry was performed using the cultured cells and mAbs directed against monocyte specific cell surface markers. In 40 all donors tested, the level of CD54 surface expression was enhanced in the presence 40 Printed from Mimosa ;WO 99/21997 of A39R, but not in the presence of heat inactivated A39R Similarly, in cultures containing control proteins CD54 surface expression was not enhanced CD54, also known as ICAM-1, is an adhesion molecule whose increased expression is considered to be indicative of cellular activation These data indicate that promoting the interaction of A39R with its receptor can activate freshly isolated 10 human monocytes Example 18 Cytokine Induction from Freshly Isolated Human Monocytes Freshly isolated human monocytes were punfied as descnbed in Example 16, 15 and cultured as descnbed in Example 17 After the overnight incubation with A39R/polyHis, monocyte supematants were examined for the presence of proinflammatory cytokines In all donors tested, IL-6 and EL-8 was induced by A39R piotein Heat inactivated A39R and control proteins did not inducted IL-6 or IL-8. Additionally, cytokine production was blocked by the inclusion of a mAb directed 20 against A39R.

The results of this expenment demonstrate that A39R, or homologues of this protein, interacting with its receptor, can induce cytokine production by freshly isolated monocytes Advantageously soluble forms of VESPR can be used in inhibit the proinflammatory activity of monocytes in response to A39R or its homologues Example 19 Monocyte Aggregation Studies In order to examine human monocyte response to the interaction of a semaphonn to its receptor on monocytes, monocytes were punfied as descnbed in 30 Example 17 and an A39R/polyHIS fusion protein was prepared as descnbed in Example 2 The fusion protein and punfied, cultured monocytes were incubated Continuing the incubation for 20 hours resulted in monocyte aggregation In view of the results demonstrated in Example 17, it is suggested that the observed monocyte aggregation occurs as a lesult of CD54 upregulation However, other factors may 35 contnbute to the aggregation as well This work confirms that the semaphonn receptor of the present invention is expressed on monocytes and that the interaction between A39R and VESPR results m monocyte aggregation Similar to B cells, monocytes aggregation is indicative of their activation 40 41 Printed from Mimosa

Claims (23)

WO 99/21997 PCT/US98/22879 5 Example 20 Monoclonal Antibodies to VESPR 5 This example illustrates a method for preparing antibodies to VESPR polypeptides. Punfied VESPR polypeptide is prepared as described in Example 9. The punfied protein is used to generate antibodies against VESPR as described in 10 U.S. Patent 4,411,993. Briefly, mice are immunized at 0, 2 and 6 weeks with 10 jig with VESPR. The pnmary immunization is prepared with TITERMAX adjuvant, from Vaxcell, Inc., and subsequent immunizations are prepared with incomplete Freund's adjuvant (IFA). At 11 weeks, the mice are IV boosted with 3-4 jag VESPR in PBS. Three days after the IV boost, splenocytes are harvested and fused with an 15 Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution. Hybridoma supematants are screened for VESPR antibodies by dot blot assay against VESPR and an irrelevant Fc protein. INTELLECTUAL PROPERTY OFFICE OF N.Z. 42 3 0 JAN 2002 RECEIVED What is claimed is:

1. An isolated VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of: (a) SEQ ID NO-2; and (b) amino acids x1 to 944 of SEQ ID NO:2, wherein x1 is amino acid 1 or 35 of SEQ ID NO:2; wherein the binding protein can bind a semaphorin.

2. The VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide of claim 1, wherein the VESPR polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of 1 (a) or 1 (b).

3. The VESPR polypeptide of claim 1 wherein the semaphorin to which the VESPR polypeptide is capable of binding is selected from the group consisting of A39R semaphorin and AHV semaphorin.

4. A soluble VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence that is at least 90% identical to x1 to 944 of SEQ ID NO:2, wherein x1 is amino acid 1 or 35; and (b) a fragment of the sequence of (a); wherein the binding portion is capable of binding a semaphorin.

5. The soluble VESPR polypeptide of claim 4 wherein the amino acid sequence is identical to amino acids x1 to 944 of SEQ ID NO:2, wherein x1 is amino acid 1 or 35 of SEQ ID NO:2.

6. An isolated polynucleotide encoding a VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide, the polynucleotide selected from the group consisting of. (a) a polynucleotide of SEQ ID NO'1; (b) polynucletide sequences that encode a polypeptide that comprises a binding portion consisting of an amino acid sequence at least 80% identical to SEQ ID NO.2; (c) polynucleotide sequences that encode a polypeptide that comprises a binding portion consisting of an amino acid sequence at least 80% identical to a fragment of SEQ ID N0:2, wherein the fragment extends from amino acid x1 to 944 of SEQ ID N0:2, wherein x1 is amino acid 1 or 35 of SEQ ID NO'2; and (d) polynucleotides complementary to the polynucleotides of (a), (b), or (c); , INTELLECTUAL PROPER! , , , , , , OrFICE OF NZ wherein the binding portion can bind a semaphonn , 2 7 FEB 2002 43 received

7. An isolated polynucleotide encoding a VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide wherein the VESPR polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2.

8. The isolated polynucleotide of claim 7 wherein the VESPR polypeptide comprises the amino acid sequence of SEQ ID NO:2.

9 An isolated polynucleotide encoding a soluble VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide, wherein the soluble VESPR polypeptide comprises a binding portion consisting of an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: (a) amino acids x1 to 944 of SEQ ID NO'2, wherein x1 is ammo acid 1 or 35; and (b) a fragment of the sequence of (a); wherein the binding portion can bind a semaphorin.

10. A polynucleotide of claim 9 wherein the soluble VESPR polypeptide comprises an amino acid sequence selected from the group consisting of: (a) amino acids x1 to 944 of SEQ ID NO:2, wherein x1 is amino acid 1 or 35; and (b) a fragment of (a).

11. A fusion protein comprising a binding portion consisting of an amino acid sequence selected from the group consisting of. (a) x1 to 944 where x1 is amino acid 1 or 35 of SEQ ID NO:2; (b) a fragment of (a); and (c) an amino acid sequence that is at least 80% identical to the amino acids of (a) or (b); wherein the binding portion can bind a semaphorin.

12 A recombinant expression vector comprising the polynucleotide of claim 6.

13. A process for preparing a VESPR polypeptide, the process comprising culturing a host cell transformed with an expression vector of claim 12 under conditions that promote expression of the polypeptide, and recovering the polypeptide

14 A composition comprising a suitable diluent carrier and a polypeptide of claim 1.

15. An antibody that is immunoreactive with a polypeptide, consisting of SEQ ID NO:2

16. Use of a polypeptide of any one of claims 1-5, 22, or 23 in the manufacture of a medicament for treating inflammatory disease in a mammal. 44 INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 7 FEB 2002 RECEIVED

17. A method of screening for binding to a VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide, the method comprising contacting a mixture containing a semaphorin, or cells that express a semaphorin, with a VESPR polypeptide and detecting binding to the VESPR polypeptide, wherein the VESPR polypeptide comprises a binding portion consisting of an amino acid sequence selected from the group consisting of1 (a) SEQ ID NO:2; (b) an amino acid sequence that is at least 80% identical to SEQ ID NO-2; (c) amino acids x1 to 944 of SEQ ID NO:2, wherein x1 is amino acid 1 or 35; (d) an amino acid sequence that is at least 80% identical to the amino acid sequence of (c); and (e) fragments of (a), (b), (c), or (d), wherein the binding portion can bind a semaphorin.

18. The method of claim 17, wherein the VESPR polypeptide comprises amino acids 35 to 944 of SEQ ID NO:2.

19. The method of claim 17, wherein the VESPR polypeptide is fused to a peptide that facilitates purification and/or identification.

20. The method of claim 17, wherein the VESPR polypeptide is bound to a solid support.

21. The method of claim 17, wherein the mixture contains cells that express a semaphorin

22 The VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide of claim 1, wherein the VESPR polypeptide comprises an amino acid sequence that is identical to the amino acid sequence of 1(a) or 1(b).

23. The VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide of claim 1 consisting of an amino acid sequence that is at least 80% identical to SEQ ID NO:2. END OF CLAIMS ■INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 7 FEB 2002 received 45 WO 99/21997 PCT/US98/22879 SEQUENCE LISTING (1) GENERAL INFORMATION (1) APPLICANT Immunex Corporation, Melanxe K Spriggs, Michael R Comeau, Robert F DuBose, Richard S Johnson (ll) TITLE OF INVENTION VIRAL ENCODED SEMAPHORIN PROTEIN RECEPTOR DNA AND POLYPEPTIDES (ill) NUMBER OF SEQUENCES. 10 (lv) CORRESPONDENCE ADDRESS. (A) ADDRESSEE Jams C Henry (B) STREET. 51 University St (C) CITY- Seattle (D) STATE WA (E) COUNTRY- US (F) ZIP 98101 (v) COMPUTER READABLE FORM (A) MEDIUM TYPE Floppy disk (B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM- PC-DOS/MS-DOS (D) SOFTWARE Patentln Release #1 0, Version #1.30 (vi) CURRENT APPLICATION DATA (A) APPLICATION NUMBER --to be assigned-- (B) FILING DATE 28-OCT-98 (C) CLASSIFICATION (vil) PRIOR APPLICATION DATA (A) APPLICATION NUMBER US 08/958,598 (converted to a Provisional, see below) (B) FILING DATE 28-OCT-1997 (C) CLASSIFICATION (vi1) PRIOR APPLICATION DATA (A) APPLICATION NUMBER --to be assigned-- (USSN 08/958,598 conversion to Provisional application) (B) FILING DATE 28-OCT-1997 (C) CLASSIFICATION- (vin) ATTORNEY/AGENT INFORMATION (A) NAME: Henry, Jams C (B) REGISTRATION NUMBER 34,347 (C) REFERENCE/DOCKET NUMBER 2631-WO (IX) TELECOMMUNICATION INFORMATION (A) TELEPHONE (2 06)470-4189 (B) TELEFAX (2 06)23 3-0644 (2) INFORMATION FOR SEQ ID NO 1 1 Printed from Mimosa WO 99/21997 PCT/US98/22879 (l) SEQUENCE CHARACTERISTICS (A) LENGTH- 4707 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY linear (ll) MOLECULE TYPE CDNA (ill) HYPOTHETICAL NO (IV) ANTI-SENSE. NO (IX) FEATURE (A) NAME/KEY CDS (B) LOCATION 1 4707 (XI) SEQUENCE DESCRIPTION SEQ ID NO 1 ATG GAG GTC TCC CGG AGG AAG GCG CCG CCG CGC CCC CCG CGC CCC GCA 48 MeC Glu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala 15 10 15 GCG CCA CTG CCC CTG CTC GCC TAT CTG CTG GCA CTG GCG GCT CCC GGC 96 Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly 20 25 30 CGG GGC GCG GAC GAG CCC GTG TGG CGG TCG GAG CAA GCC ATC GGA GCC 144 Arg Gly Ala Asp Glu Pro Val Trp Arg Ser Glu Gin Ala lie Gly Ala 35 40 45 ATC GCG GCG AGC CAG GAG GAC GGC GTG TTT GTG GCG AGC GGC AGC TGC 192 lie Ala Ala Ser Gin Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys 50 55 60 CTG GAC CAG CTG GAC TAC AGC CTG GAG CAC AGC CTC TCG CGC CTG TAC 240 Leu Asp Gin Leu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr 65 70 75 80 CGG GAC CAA GCG GGC AAC TGC ACA GAG CCG GTC TCG CTG GCG CCC CCC 288 Arg Asp Gin Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro 85 90 95 GCG CGG CCC CGG CCC GGG AGC AGC TTC AGC AAG CTG CTG CTG CCC TAC 33 6 Ala Arg Pro Arg Pro Gly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr 100 105 110 CGC GAG GGG GCG GCC GGC CTC GGG GGG CTG CTG CTC ACC GGC TGG ACC 384 Arg Glu Gly Ala Ala Gly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr 115 120 125 TTC GAC CGG GGC GCC TGC GAG GTG CGG CCC CTG GGC AAC CTG AGC CGC 432 Phe Asp Arg Gly Ala Cys Glu Val Arg Pro Leu Gly Asn Leu Ser Arg 130 135 140 AAC TCC CTG CGC AAC GGC ACC GAG GTG GTG TCG TGC CAC CCG CAG GGC 480 Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro Gin Gly 2 Printed from Mimosa -WO 99/21997 PCT/US98/22879 145 150 155 160 TCG ACG GCC GGC GTG GTG TAC CGC GCG GGC CGG AAC AAC CGC TGG TAC 528 Ser Thr Ala Gly Val Val Tyr Arg Ala Gly Arg Asn Asn Arg Trp Tyr 165 170 175 CTG GCG GTG GCC GCC ACC TAC GTG CTG CCT GAG CCG GAG ACG GCG AGC 57 6 Leu Ala Val Ala Ala Thr Tyr Val Leu Pro Glu Pro Glu Thr Ala Ser 180 185 190 CGC TGC AAC CCC GCG GCA TCC GAC CAC GAC ACG GCC ATC GCG CTC AAG 624 Arg Cys Asn Pro Ala Ala Ser Asp His Asp Thr Ala lie Ala Leu Lys 195 200 205 GAC ACG GAG GGG CGC AGC CTG GCC ACG CAG GAG CTG GGG CGC CTC AAG 672 Asp Thr Glu Gly Arg Ser Leu Ala Thr Gin Glu Leu Gly Arg Leu Lys 210 215 220 CTG TGC GAG GGC GCG GGC AGC CTG CAC TTC GTG GAC GCC TTT CTC TGG 72 0 Leu Cys Glu Gly Ala Gly Ser Leu His Phe Val Asp Ala Phe Leu Trp 225 230 235 240 AAC GGC AGC ATC TAC TTC CCC TAC TAC CCC TAC AAC TAT ACG AGC GGC 7 68 Asn Gly Ser lie Tyr Phe Pro Tyr Tyr Pro Tyr Asn Tyr Thr Ser Gly 245 250 255 GCT GCC ACC GGC TGG CCC AGC ATG GCG CGC ATC GCG CAG AGC ACC GAG 816 Ala Ala Thr Gly Trp Pro Ser Met Ala Arg lie Ala Gin Ser Thr Glu 260 265 270 GTG CTG TTC CAG GGC CAG GCA TCC CTC GAC TGC GGC CAC GGC CAC CCC 864 Val Leu Phe Gin Gly Gin Ala Ser Leu Asp Cys Gly His Gly His Pro 275 280 285 GAC GGC CGC CGC CTG CTC CTC TCC TCC AGC CTA GTG GAG GCC CTG GAC 912 Asp Gly Arg Arg Leu Leu Leu Ser Ser Ser Leu Val Glu Ala Leu Asp 290 295 300 GTC TGG GCG GGA GTG TTC AGC GCG GCC GCT GGA GAG GGC CAG GAG CGG 9 60 Val Trp Ala Gly Val Phe Ser Ala Ala Ala Gly Glu Gly Gin Glu Arg 305 310 315 320 CGC TCC CCC ACC ACC ACG GCG CTC TGC CTC TTC AGA ATG AGT GAG ATC 1008 Arg Ser Pro Thr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser Glu lie 325 330 335 CAG GCG CGC GCC AAG AGG GTC AGC TGG GAC TTC AAG ACG GCC GAG AGC 1056 Gin Ala Arg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala Glu Ser 340 345 350 CAC TGC AAA GAA GGG GAT CAA CCT GAA AGA GTC CAA CCA ATC GCA TCA 1104 His Cys Lys Glu Gly Asp Gin Pro Glu Arg Val Gin Pro lie Ala Ser 355 360 365 TCT ACC TTG ATC CAT TCC GAC CTG ACA TCC GTT TAT GGC ACC GTG GTA 1152 Ser Thr Leu lie His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val 370 375 380 3 Printed from Mimosa WO 99/21997 PCT/US98/22879 ATG AAC AGG ACT GTT TTA TTC TTG GGG ACT GGA GAT GGC CAG TTA CTT 12 00 Met Asn Arg Thr Val Leu Phe Leu Gly Thr Gly Asp Gly Gin Leu Leu 385 390 395 400 AAG GTT ATT CTT GGT GAG AAT TTG ACT TCA AAT TGT CCA GAG GTT ATC 1248 Lys Val lie Leu Gly Glu Asn Leu Thr Ser Asn Cys Pro Glu Val lie 405 410 415 TAT GAA ATT AAA GAA GAG ACA CCT GTT TTC TAC AAA CTC GTT CCT GAT 1296 Tyr Glu lie Lys Glu Glu Thr Pro Val Phe Tyr Lys Leu Val Pro Asp 420 425 430 CCT GTG AAG AAT ATC TAC ATT TAT CTA ACA GCT GGG AAA GAG GTG AGG 1344 Pro Val Lys Asn lie Tyr lie Tyr Leu Thr Ala Gly Lys Glu Val Arg 435 440 445 AGA ATT CGT GTT GCA AAC TGC AAT AAA CAT AAA TCC TGT TCG GAG TGT 1392 Arg lie Arg Val Ala Asn Cys Asn Lys His Lys Ser Cys Ser Glu Cys 450 455 460 TTA ACA GCC ACA GAC CCT CAC TGC GGT TGG TGC CAT TCG CTA CAA AGG 1440 Leu Thr Ala Thr Asp Pro His Cys Gly Trp Cys His Ser Leu Gin Arg 465 470 475 480 TGC ACT TTT CAA GGA GAT TGT GTA CAT TCA GAG AAC TTA GAA AAC TGG 1488 Cys Thr Phe Gin Gly Asp Cys Val His Ser Glu Asn Leu Glu Asn Trp 485 490 495 CTG GAT ATT TCG TCT GGA GCA AAA AAG TGC CCT AAA ATT CAG ATA ATT 1536 Leu Asp lie Ser Ser Gly Ala Lys Lys Cys Pro Lys lie Gin lie lie 500 505 510 CGA AGC AGT AAA GAA AAG ACT ACA GTG ACT ATG GTG GGA AGC TTC TCT 1584 Arg Ser Ser Lys Glu Lys Thr Thr Val Thr Met Val Gly Ser Phe Ser 515 520 525 CCA AGA CAC TCA AAG TGC ATG GTG AAG AAT GTG GAC TCT AGC AGG GAG 1632 Pro Arg His Ser Lys Cys Met Val Lys Asn Val Asp Ser Ser Arg Glu 530 535 540 CTC TGC CAG AAT AAA AGT CAG CCC AAC CGG ACC TGC ACC TGT AGC ATC 1680 Leu Cys Gin Asn Lys Ser Gin Pro Asn Arg Thr Cys Thr Cys Ser lie 545 550 555 560 CCA ACC AGA GCA ACC TAC AAA GAT GTT TCA GTT GTC AAC GTG ATG TTC 1728 Pro Thr Arg Ala Thr Tyr Lys Asp Val Ser Val Val Asn Val Met Phe 565 570 575 TCC TTC GGT TCT TGG AAT TTA TCA GAC AGA TTC AAC TTT ACC AAC TGC 177 6 Ser Phe Gly Ser Trp Asn Leu Ser Asp Arg Phe Asn Phe Thr Asn Cys 580 585 590 TCA TCA TTA AAA GAA TGC CCA GCA TGC GTA GAA ACT GGC TGC GCG TGG 1824 Ser Ser Leu Lys Glu Cys Pro Ala Cys Val Glu Thr Gly Cys Ala Trp 595 600 605 TGT AAA AGT GCA AGA AGG TGT ATC CAC CCC TTC ACA GCT TGC GAC CCT 1872 Cys Lys Ser Ala Arg Arg Cys lie His Pro Phe Thr Ala Cys Asp Pro 4 Printed from Mimosa :WO 99/21997 PCT/US98/22879 610 615 620 TCT GAT TAT GAG AGA AAC CAG GAA CAG TGT CCA GTG GCT GTC GAG AAG 192 0 Ser Asp Tyr Glu Arg Asn Gin Glu Gin Cys Pro Val Ala Val Glu Lys 625 630 635 640 ACA TCA GGA GGA GGA AGA CCC AAG GAG AAC AAG GGG AAC AGA ACC AAC 1968 Thr Ser Gly Gly Gly Arg Pro Lys Glu Asn Lys Gly Asn Arg Thr Asn 645 650 655 CAG GCT TTA CAG GTC TTC TAC ATT AAG TCC ATT GAG CCA CAG AAA GTA 2016 Gin Ala Leu Gin Val Phe Tyr lie Lys Ser lie Glu Pro Gin Lys Val 660 665 670 TCG ACA TTA GGG AAA AGC AAC GTG ATA GTA ACG GGA GCA AAC TTT ACC 2 064 Ser Thr Leu Gly Lys Ser Asn Val lie Val Thr Gly Ala Asn Phe Thr 675 680 685 CGG GCA TCG AAC ATC ACA ATG ATC CTG AAA GGA ACC AGT ACC TGT GAT 2112 Arg Ala Ser Asn lie Thr Met lie Leu Lys Gly Thr Ser Thr Cys Asp 690 695 700 AAG GAT GTG ATA CAG GTT AGC CAT GTG CTA AAT GAC ACC CAC ATG AAA 2160 Lys Asp Val lie Gin Val Ser His Val Leu Asn Asp Thr His Met Lys 705 710 715 720 TTC TCT CTT CCA TCA AGC CGG AAA GAA ATG AAG GAT GTG TGT ATC CAG 2208 Phe Ser Leu Pro Ser Ser Arg Lys Glu Met Lys Asp Val Cys lie Gin 725 730 735 TTT GAT GGT GGG AAC TGC TCT TCT GTG GGA TCC TTA TCC TAC ATT GCT 22 56 Phe Asp Gly Gly Asn Cys Ser Ser Val Gly Ser Leu Ser Tyr lie Ala 740 745 750 CTG CCA CAT TGT TCC CTT ATA TTT CCT GCT ACC ACC TGG ATC AGT GGT 2304 Leu Pro His Cys Ser Leu lie Phe Pro Ala Thr Thr Trp lie Ser Gly 755 760 765 GGT CAA AAT ATA ACC ATG ATG GGC AGA AAT TTT GAT GTA ATT GAC AAC 2352 Gly Gin Asn lie Thr Met Met Gly Arg Asn Phe Asp Val lie Asp Asn 770 775 780 TTA ATC ATT TCA CAT GAA TTA AAA GGA AAC ATA AAT GTC TCT GAA TAT 2400 Leu lie He Ser His Glu Leu Lys Gly Asn lie Asn Val Ser Glu Tyr 785 790 795 800 TGT GTG GCG ACT TAC TGC GGG TTT TTA GCC CCC AGT TTA AAG AGT TCA 2448 Cys Val Ala Thr Tyr Cys Gly Phe Leu Ala Pro Ser Leu Lys Ser Ser 805 810 815 AAA GTG CGC ACG AAT GTC ACT GTG AAG CTG AGA GTA CAA GAC ACC TAC 2496 Lys Val Arg Thr Asn Val Thr Val Lys Leu Arg Val Gin Asp Thr Tyr 820 825 830 TTG GAT TGT GGA ACC CTG CAG TAT CGG GAG GAC CCC AGA TTC ACG GGG 2 544 Leu Asp Cys Gly Thr Leu Gin Tyr Arg Glu Asp Pro Arg Phe Thr Gly 835 840 845 5 Printed from Mimosa WO 99/21997 PCT/US98/22879 TAT CGG GTG GAA TCC GAG GTG GAC ACA GAA CTG GAA GTG AAA ATT CAA 2592 Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val Lys lie Gin 850 855 860 AAA GAA AAT GAC AAC TTC AAT ATT TCC AAA AAA GAC ATT GAA ATT ACT 2 640 Lys Glu Asn Asp Asn Phe Asn lie Ser Lys Lys Asp lie Glu lie Thr 865 870 875 880 CTC TTC CAT GGG GAA AAT GGG CAA TTA AAT TGC AGT TTT GAA AAT ATT 2 688 Leu Phe His Gly Glu Asn Gly Gin Leu Asn Cys Ser Phe Glu Asn lie 885 890 895 ACT AGA AAT CAA GAT CTT ACC ACC ATC CTT TGC AAA ATT AAA GGC ATC 273 6 Thr Arg Asn Gin Asp Leu Thr Thr lie Leu Cys Lys lie Lys Gly lie 900 905 910 AAG ACT GCA AGC ACC ATT GCC AAC TCT TCT AAG AAA GTT CGG GTC AAG 2784 Lys Thr Ala Ser Thr lie Ala Asn Ser Ser Lys Lys Val Arg Val Lys 915 920 925 CTG GGA AAC CTG GAG CTC TAC GTC GAG CAG GAG TCA GTT CCT TCC ACA 2832 Leu Gly Asn Leu Glu Leu Tyr Val Glu Gin Glu Ser Val Pro Ser Thr 930 935 940 TGG TAT TTT CTG ATT GTG CTC CCT GTC TTG CTA GTG ATT GTC ATT TTT 2 880 Trp Tyr Phe Leu lie Val Leu Pro Val Leu Leu Val lie Val lie Phe 945 950 955 960 GCG GCC GTG GGG GTG ACC AGG CAC AAA TCG AAG GAG CTG AGT CGC AAA 2 92 8 Ala Ala Val Gly Val Thr Arg His Lys Ser Lys Glu Leu Ser Arg Lys 965 970 975 CAG AGT CAA CAA CTA GAA TTG CTG GAA AGC GAG CTC CGG AAA GAG ATA 2 97 6 Gin Ser Gin Gin Leu Glu Leu Leu Glu Ser Glu Leu Arg Lys Glu lie 980 985 990 CGT GAC GGC TTT GCT GAG CTG CAG ATG GAT AAA TTG GAT GTG GTT GAT 3024 Arg Asp Gly Phe Ala Glu Leu Gin Met Asp Lys Leu Asp Val Val Asp 995 1000 1005 AGT TTT GGA ACT GTT CCC TTC CTT GAC TAC AAA CAT TTT GCT CTG AGA 3072 Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg 1010 1015 1020 ACT TTC TTC CCT GAG TCA GGT GGC TTC ACC CAC ATC TTC ACT GAA GAT 3120 Thr Phe Phe Pro Glu Ser Gly Gly Phe Thr His He Phe Thr Glu Asp 1025 1030 1035 1040 ATG CAT AAC AGA GAC GCC AAC GAC AAG AAT GAA AGT CTC ACA GCT TTG 3168 Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu 1045 1050 1055 GAT GCC CTA ATC TGT AAT AAA AGC TTT CTT GTT ACT GTC ATC CAC ACC 3216 Asp Ala Leu He Cys Asn Lys Ser Phe Leu Val Thr Val lie His Thr 1060 1065 1070 CTT GAA AAG CAG AAG AAC TTT TCT GTG AAG GAC AGG TGT CTG TTT GCC 3264 Leu Glu Lys Gin Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Ala 6 Printed from Mimosa WO 99/21997 PCT/US98/22879 1075 1080 1085 TCC TTC CTA ACC ATT GCA CTG CAA ACC AAG CTG GTC TAC CTG ACC AGC 3312 Ser Phe Leu Thr lie Ala Leu Gin Thr Lys Leu Val Tyr Leu Thr Ser 1090 1095 1100 ATC CTA GAG GTG CTG ACC AGG GAC TTG ATG GAA CAG TGT AGT AAC ATG 3360 lie Leu Glu Val Leu Thr Arg Asp Leu Met Glu Gin Cys Ser Asn Met 1105 1110 1115 1120 CAG CCG AAA CTC ATG CTG AGA CGC ACG GAG TCC GTC GTC GAA AAA CTC 3408 Gin Pro Lys Leu Met Leu Arg Arg Thr Glu Ser Val Val Glu Lys Leu 1125 1130 1135 CTC ACA AAC TGG ATG TCC GTC TGC CTT TCT GGA TTT CTC CGG GAG ACT 3456 Leu Thr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr 1140 1145 1150 GTC GGA GAG CCC TTC TAT TTG CTG GTG ACG ACT CTG AAC CAG AAA ATT 3504 Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gin Lys lie 1155 1160 1165 AAC AAG GGT CCC GTG GAT GTA ATC ACT TGC AAA GCC CTG TAC ACA CTT 3 552 Asn Lys Gly Pro Val Asp Val lie Thr Cys Lys Ala Leu Tyr Thr Leu 1170 1175 1180 AAT GAA GAC TGG CTG TTG TGG CAG GTT CCG GAA TTC AGT ACT GTG GCA 3600 Asn Glu Asp Trp Leu Leu Trp Gin Val Pro Glu Phe Ser Thr Val Ala 1185 1190 1195 1200 TTA AAC GTC GTC TTT GAA AAA ATC CCG GAA AAC GAG AGT GCA GAT GTC 3 648 Leu Asn Val Val Phe Glu Lys lie Pro Glu Asn Glu Ser Ala Asp Val 1205 1210 1215 TGT CGG AAT ATT TCA GTC AAT GTT CTC GAC TGT GAC ACC ATT GGC CAA 3 696 Cys Arg Asn lie Ser Val Asn Val Leu Asp Cys Asp Thr lie Gly Gin 1220 1225 1230 GCC AAA GAA AAG ATT TTC CAA GCA TTC TTA AGC AAA AAT GGC TCT CCT 3744 Ala Lys Glu Lys lie Phe Gin Ala Phe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 TAT GGA CTT CAG CTT AAT GAA ATT GGT CTT GAG CTT CAA ATG GGC ACA 3792 Tyr Gly Leu Gin Leu Asn Glu lie Gly Leu Glu Leu Gin Met Gly Thr 1250 1255 1260 CGA CAG AAA GAA CTT CTG GAC ATC GAC AGT TCC TCC GTG ATT CTT GAA 3840 Arg Gin Lys Glu Leu Leu Asp lie Asp Ser Ser Ser Val lie Leu Glu 1265 1270 1275 1280 GAT GGA ATC ACC AAG CTA AAC ACC ATT GGC CAC TAT GAG ATA TCA AAT 3 888 Asp Gly lie Thr Lys Leu Asn Thr lie Gly His Tyr Glu lie Ser Asn 1285 1290 1295 GGA TCC ACT ATA AAA GTC TTT AAG AAG ATA GCA AAT TTT ACT TCA GAT 3 93 6 Gly Ser Thr lie Lys Val Phe Lys Lys lie Ala Asn Phe Thr Ser Asp 1300 1305 1310 7 Printed from Mimosa WO 99/21997 PCT/US98/22879 GTG GAG TAC TCG GAT GAC CAC TGC CAT TTG ATT TTA CCA GAT TCG GAA 3 984 . Val Glu Tyr Ser Asp Asp His Cys His Leu lie Leu Pro Asp Ser Glu 1315 1320 1325 GCA TTC CAA GAT GTG CAA GGA AAG AGA CAT CGA GGG AAG CAC AAG TTC 4032 Ala Phe Gin Asp Val Gin Gly Lys Arg His Arg Gly Lys His Lys Phe 1330 1335 1340 AAA GTA AAA GAA ATG TAT CTG ACA AAG CTG CTG TCG ACC AAG GTG GCA 4080 Lys Val Lys Glu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala 1345 1350 1355 1360 ATT CAT TCT GTG CTT GAA AAA CTT TTT AGA AGC ATT TGG AGT TTA CCC 412 8 lie His Ser Val Leu Glu Lys Leu Phe Arg Ser lie Trp Ser Leu Pro 1365 1370 1375 AAC AGC AGA GCT CCA TTT GCT ATA AAA TAC TTT TTT GAC TTT TTG GAC 4176 Asn Ser Arg Ala Pro Phe Ala lie Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 GCC CAG GCT GAA AAC AAA AAA ATC ACA GAT CCT GAC GTC GTA CAT ATT 4224 Ala Gin Ala Glu Asn Lys Lys lie Thr Asp Pro Asp Val Val His lie 1395 1400 1405 TGG AAA ACA AAC AGC CTT CCT CTT CGC TTC TGG GTA AAC ATC CTG AAG 4272 Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn lie Leu Lys 1410 1415 1420 AAC CCT CAG TTT GTC TTT GAC ATT AAG AAG ACA CCA CAT ATA GAC GGC 432 0 Asn Pro Gin Phe Val Phe Asp lie Lys Lys Thr Pro His lie Asp Gly 1425 1430 1435 1440 TGT TTG TCA GTG ATT GCC CAG GCA TTC ATG GAT GCA TTT TCT CTC ACA 43 6 8 Cys Leu Ser Val lie Ala Gin Ala Phe Met Asp Ala Phe Ser Leu Thr 1445 1450 1455 GAG CAG CAA CTA GGG AAG GAA GCA CCA ACT AAT AAG CTT CTC TAT GCC 4416 Glu Gin Gin Leu Gly Lys Glu Ala Pro Thr Asn Lys Leu Leu Tyr Ala 1460 1465 1470 AAG GAT ATC CCA ACC TAC AAA GAA GAA GTA AAA TCT TAT TAC AAA GCA 4464 Lys Asp lie Pro Thr Tyr Lys Glu Glu Val Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 ATC AGG GAT TTG CCT CCA TTG TCA TCC TCA GAA ATG GAA GAA TTT TTA 4512 lie Arg Asp Leu Pro Pro Leu Ser Ser Ser Glu Met Glu Glu Phe Leu 1490 1495 1500 ACT CAG GAA TCT AAG AAA CAT GAA AAT GAA TTT AAT GAA GAA GTG GCC 4560 Thr Gin Glu Ser Lys Lys His Glu Asn Glu Phe Asn Glu Glu Val Ala 1505 1510 1515 1520 TTG ACA GAA ATT TAC AAA TAC ATC GTA AAA TAT TTT GAT GAG ATT CTA 4608 Leu Thr Glu lie Tyr Lys Tyr lie Val Lys Tyr Phe Asp Glu lie Leu 1525 1530 1535 AAT AAA CTA GAA AGA GAA CGA GGG CTG GAA GAA GCT CAG AAA CAA CTC 4656 Asn Lys Leu Glu Arg Glu Arg Gly Leu Glu Glu Ala Gin Lys Gin Leu Printed from Mimosa WO 99/21997 PCT/US98/22879 1540 1545 1550 TTG CAT GTA AAA GTC TTA TTT GAT GAA AAG AAG AAA TGC AAG TGG ATG 4704 Leu His Val Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys Trp Met 1555 1560 1565 TAA 4707 (2) INFORMATION FOR SEQ ID NO 2 {i) SEQUENCE CHARACTERISTICS (A) LENGTH 1569 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (11) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION SEQ ID NO 2 Met Glu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala 15 10 15 Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly 20 25 30 Arg Gly Ala Asp Glu Pro Val Trp Arg Ser Glu Gin Ala lie Gly Ala 35 40 45 lie Ala Ala Ser Gin Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys 50 55 60 Leu Asp Gin Leu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr 65 70 75 80 Arg Asp Gin Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro 85 90 95 Ala Arg Pro Arg Pro Gly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr 100 105 110 Arg Glu Gly Ala Ala Gly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr 115 120 125 Phe Asp Arg Gly Ala Cys Glu Val Arg Pro Leu Gly Asn Leu Ser Arg 130 135 140 Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro Gin Gly 145 150 155 160 Ser Thr Ala Gly Val Val Tyr Arg Ala Gly Arg Asn Asn Arg Trp Tyr 165 170 175 Leu Ala Val Ala Ala Thr Tyr Val Leu Pro Glu Pro Glu Thr Ala Ser 180 185 190 Arg Cys Asn Pro Ala Ala Ser Asp His Asp Thr Ala lie Ala Leu Lys Printed from Mimosa WO 99/21997 PCT/US98/22879 195 200 205 Asp Thr Glu Gly Arg Ser Leu Ala Thr Gin Glu Leu Gly Arg Leu Lys 210 215 220 Leu Cys Glu Gly Ala Gly Ser Leu His Phe Val Asp Ala Phe Leu Trp 225 230 235 240 Asn Gly Ser lie Tyr Phe Pro Tyr Tyr Pro Tyr Asn Tyr Thr Ser Gly 245 250 255 Ala Ala Thr Gly Trp Pro Ser Met Ala Arg lie Ala Gin Ser Thr Glu 260 265 270 Val Leu Phe Gin Gly Gin Ala Ser Leu Asp Cys Gly His Gly His Pro 275 280 285 Asp Gly Arg Arg Leu Leu Leu Ser Ser Ser Leu Val Glu Ala Leu Asp 290 295 300 Val Trp Ala Gly Val Phe Ser Ala Ala Ala Gly Glu Gly Gin Glu Arg 305 310 315 320 Arg Ser Pro Thr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser Glu lie 325 330 335 Gin Ala Arg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala Glu Ser 340 345 350 His Cys Lys Glu Gly Asp Gin Pro Glu Arg Val Gin Pro lie Ala Ser 355 360 365 Ser Thr Leu lie His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val 370 375 380 Met Asn Arg Thr Val Leu Phe Leu Gly Thr Gly Asp Gly Gin Leu Leu 385 390 395 400 Lys Val lie Leu Gly Glu Asn Leu Thr Ser Asn Cys Pro Glu Val lie 405 410 415 Tyr Glu lie Lys Glu Glu Thr Pro Val Phe Tyr Lys Leu Val Pro Asp 420 425 430 Pro Val Lys Asn lie Tyr lie Tyr Leu Thr Ala Gly Lys Glu Val Arg 435 440 445 Arg lie Arg Val Ala Asn Cys Asn Lys His Lys Ser Cys Ser Glu Cys 450 455 460 Leu Thr Ala Thr Asp Pro His Cys Gly Trp Cys His Ser Leu Gin Arg 465 470 475 480 Cys Thr Phe Gin Gly Asp Cys Val His Ser Glu Asn Leu Glu Asn Trp 485 490 495 Leu Asp lie Ser Ser Gly Ala Lys Lys Cys Pro Lys lie Gin lie lie 500 505 510 10 Printed from Mimosa :WO 99/21997 PCT/US98/22879 Arg Ser Ser Lys Glu Lys Thr Thr Val Thr Met Val Gly Ser Phe Ser 515 520 525 Pro Arg His Ser Lys Cys Met Val Lys Asn Val Asp Ser Ser Arg Glu 530 535 540 Leu Cys Gin Asn Lys Ser Gin Pro Asn Arg Thr Cys Thr Cys Ser lie 545 550 555 560 Pro Thr Arg Ala Thr Tyr Lys Asp Val Ser Val Val Asn Val Met Phe 565 570 575 Ser Phe Gly Ser Trp Asn Leu Ser Asp Arg Phe Asn Phe Thr Asn Cys 580 585 590 Ser Ser Leu Lys Glu Cys Pro Ala Cys Val Glu Thr Gly Cys Ala Trp 595 600 605 Cys Lys Ser Ala Arg Arg Cys lie His Pro Phe Thr Ala Cys Asp Pro 610 615 620 Ser Asp Tyr Glu Arg Asn Gin Glu Gin Cys Pro Val Ala Val Glu Lys 625 630 635 640 Thr Ser Gly Gly Gly Arg Pro Lys Glu Asn Lys Gly Asn Arg Thr Asn 645 650 655 Gin Ala Leu Gin Val Phe Tyr lie Lys Ser lie Glu Pro Gin Lys Val 660 665 670 Ser Thr Leu Gly Lys Ser Asn Val lie Val Thr Gly Ala Asn Phe Thr 675 680 685 Arg Ala Ser Asn lie Thr Met lie Leu Lys Gly Thr Ser Thr Cys Asp 690 695 700 Lys Asp Val lie Gin Val Ser His Val Leu Asn Asp Thr His Met Lys 705 710 715 720 Phe Ser Leu Pro Ser Ser Arg Lys Glu Met Lys Asp Val Cys lie Gin 725 730 735 Phe Asp Gly Gly Asn Cys Ser Ser Val Gly Ser Leu Ser Tyr lie Ala 740 745 750 Leu Pro His Cys Ser Leu lie Phe Pro Ala Thr Thr Trp lie Ser Gly 755 760 765 Gly Gin Asn lie Thr Met Met Gly Arg Asn Phe Asp Val lie Asp Asn 770 775 780 Leu He lie Ser His Glu Leu Lys Gly Asn lie Asn Val Ser Glu Tyr 785 790 795 800 Cys Val Ala Thr Tyr Cys Gly Phe Leu Ala Pro Ser Leu Lys Ser Ser 805 810 815 11 Printed from Mimosa .WO 99/21997 PCT/US98/22879 Lys Val Arg Thr Asn Val Thr Val Lys Leu Arg Val Gin Asp Thr Tyr 820 825 830 Leu Asp Cys Gly Thr Leu Gin Tyr Arg Glu Asp Pro Arg Phe Thr Gly 835 840 845 Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val Lys lie Gin 850 855 860 Lys Glu Asn Asp Asn Phe Asn lie Ser Lys Lys Asp lie Glu lie Thr 865 870 875 880 Leu Phe His Gly Glu Asn Gly Gin Leu Asn Cys Ser Phe Glu Asn lie 885 890 895 Thr Arg Asn Gin Asp Leu Thr Thr lie Leu Cys Lys lie Lys Gly lie 900 905 910 Lys Thr Ala Ser Thr lie Ala Asn Ser Ser Lys Lys Val Arg Val Lys 915 920 925 Leu Gly Asn Leu Glu Leu Tyr Val Glu Gin Glu Ser Val Pro Ser Thr 930 935 940 Trp Tyr Phe Leu lie Val Leu Pro Val Leu Leu Val lie Val lie Phe 945 950 955 960 Ala Ala Val Gly Val Thr Arg His Lys Ser Lys Glu Leu Ser Arg Lys 965 970 975 Gin Ser Gin Gin Leu Glu Leu Leu Glu Ser Glu Leu Arg Lys Glu lie 980 985 990 Arg Asp Gly Phe Ala Glu Leu Gin Met Asp Lys Leu Asp Val Val Asp 995 1000 1005 Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg 1010 1015 1020 Thr Phe Phe Pro Glu Ser Gly Gly Phe Thr His lie Phe Thr Glu Asp 1025 1030 1035 1040 Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu 1045 1050 1055 Asp Ala Leu lie Cys Asn Lys Ser Phe Leu Val Thr Val lie His Thr 1060 1065 1070 Leu Glu Lys Gin Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Ala 1075 1080 1085 Ser Phe Leu Thr lie Ala Leu Gin Thr Lys Leu Val Tyr Leu Thr Ser 1090 1095 1100 lie Leu Glu Val Leu Thr Arg Asp Leu Met Glu Gin Cys Ser Asn Met 1105 1110 1115 1120 12 Printed from Mimosa WO 99/21997 PCT/US98/22879 Gin Pro Lys Leu Met Leu Arg Arg Thr Glu Ser Val Val Glu Lys Leu 1125 1130 1135 Leu Thr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr 1140 1145 1150 Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gin Lys lie 1155 1160 1165 Asn Lys Gly Pro Val Asp Val lie Thr Cys Lys Ala Leu Tyr Thr Leu 1170 1175 1180 Asn Glu Asp Trp Leu Leu Trp Gin Val Pro Glu Phe Ser Thr Val Ala 1185 1190 1195 1200 Leu Asn Val Val Phe Glu Lys lie Pro Glu Asn Glu Ser Ala Asp Val 1205 1210 1215 Cys Arg Asn lie Ser Val Asn Val Leu Asp Cys Asp Thr lie Gly Gin 1220 1225 1230 Ala Lys Glu Lys lie Phe Gin Ala Phe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 Tyr Gly Leu Gin Leu Asn Glu lie Gly Leu Glu Leu Gin Met Gly Thr 1250 1255 1260 Arg Gin Lys Glu Leu Leu Asp lie Asp Ser Ser Ser Val lie Leu Glu 1265 1270 1275 1280 Asp Gly lie Thr Lys Leu Asn Thr lie Gly His Tyr Glu lie Ser Asn 1285 1290 1295 Gly Ser Thr lie Lys Val Phe Lys Lys lie Ala Asn Phe Thr Ser Asp 1300 1305 1310 Val Glu Tyr Ser Asp Asp His Cys His Leu lie Leu Pro Asp Ser Glu 1315 1320 1325 Ala Phe Gin Asp Val Gin Gly Lys Arg His Arg Gly Lys His Lys Phe 1330 1335 1340 Lys Val Lys Glu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala 1345 1350 1355 1360 lie His Ser Val Leu Glu Lys Leu Phe Arg Ser lie Trp Ser Leu Pro 1365 1370 1375 Asn Ser Arg Ala Pro Phe Ala lie Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 Ala Gin Ala Glu Asn Lys Lys lie Thr Asp Pro Asp Val Val His He 1395 1400 1405 Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn lie Leu Lys 1410 1415 1420 13 Printed from Mimosa - WO 99/21997 PCT/US98/22879 Asn Pro Gin Phe Val Phe Asp lie Lys Lys Thr Pro His lie Asp Gly 1425 1430 1435 1440 Cys Leu Ser Val lie Ala Gin Ala Phe Met Asp Ala Phe Ser Leu Thr 1445 1450 1455 Glu Gin Gin Leu Gly Lys Glu Ala Pro Thr Asn Lys Leu Leu Tyr Ala 1460 1465 1470 Lys Asp lie Pro Thr Tyr Lys Glu Glu Val Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 lie Arg Asp Leu Pro Pro Leu Ser Ser Ser Glu Met Glu Glu Phe Leu 1490 1495 1500 Thr Gin Glu Ser Lys Lys His Glu Asn Glu Phe Asn Glu Glu Val Ala 1505 1510 1515 1520 Leu Thr Glu lie Tyr Lys Tyr lie Val Lys Tyr Phe Asp Glu lie Leu 1525 1530 1535 Asn Lys Leu Glu Arg Glu Arg Gly Leu Glu Glu Ala Gin Lys Gin Leu 1540 1545 1550 Leu His Val Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys Trp Met 1555 1560 1565 (2) INFORMATION FOR SEQ ID NO 3 (1) SEQUENCE CHARACTERISTICS (A) LENGTH 31 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ll) MOLECULE TYPE CDNA (xi) SEQUENCE DESCRIPTION SEQ ID NO 3 TGTCACTAGT ATCGAATGGC ATAAGTTTGA A 31 (2) INFORMATION FOR SEQ ID NO•4 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 3 3 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY linear (11) MOLECULE TYPE CDNA (ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO 14 Printed from Mimosa WO 99/21997 PCT/US98/22879 (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 GACAGCGGCC GCCTATTACA TTTTAAGTAT TTT 3 3 (2) INFORMATION FOR SEQ ID NO 5- (l) SEQUENCE CHARACTERISTICS (A) LENGTH 18 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE primer (ill) HYPOTHETICAL NO (IV) ANTI-SENSE NO (IX) FEATURE (A) NAME/KEY (B) LOCATION (XI) SEQUENCE DESCRIPTION SEQ ID NO 5 GCGGGACTCA GAGTCACC 18 (2) INFORMATION FOR SEQ ID NO.6 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 43 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE primer ' (ill) HYPOTHETICAL NO (IV) ANTI-SENSE NO (XI) SEQUENCE DESCRIPTION SEQ ID NO.6 GGATCCTAAT ACGACTCACT ATAGGGAGGA AACCACTCCG AAC 43 (2) INFORMATION FOR SEQ ID NO.7 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 1983 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear 15 Printed from Mimosa WO 99/21997 PCT/US98/22879 (11) MOLECULE TYPE CDNA (ill) HYPOTHETICAL NO (IV) ANTI-SENSE NO (IX) FEATURE (A) NAME/KEY CDS (B) LOCATION 1 1983 (xi) SEQUENCE DESCRIPTION SEQ ID NO 7 ATG TTC CAT GTT TCT TTT AGA TAT ATC TTT GGA ATT CCT CCA CTG ATC 48 Met Phe His Val Ser Phe Arg Tyr lie Phe Gly lie Pro Pro Leu lie 15 10 15 CTT GTT CTG CTG CCT GTC ACT AGC TCT GAC TAC AAA GAT GAC GAT GAT 96 Leu Val Leu Leu Pro Val Thr Ser Ser Asp Tyr Lys Asp Asp Asp Asp 20 25 30 AAA AGA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT 144 Lys Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 35 40 45 GAA GCC GAG GGC GCG CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG 192 Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 50 55 60 GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG 24 0 Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 65 70 75 80 GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC 2 88 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 85 ' 90 95 GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC 33 6 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 100 105 110 AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC 3 84 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 115 120 125 TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC 432 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 130 135 140 CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA 480 Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 145 150 155 160 GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG 52 8 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 165 170 175 16 Printed from Mimosa WO 99/21997 PCT/US98/22879 AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 180 185 190 576 ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 195 200 205 624 ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAT AGC Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 210 215 220 672 AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 225 230 235 240 720 TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 245 250 255 768 CTC TCC CTG TCT CCG GGT AAA GGA GGG GGC GGA TCA GGG GGC GGA GGA Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 260 265 270 816 TCT ACT AGT ATC GAA TGG CAT AAG TTT GAA ACG AGT GAA GAA ATA ATT Ser Thr Ser lie Glu Trp His Lys Phe Glu Thr Ser Glu Glu lie lie 275 280 285 864 TCT ACT TAC TTA ATA GAT GAT GTA TTA TAC ACG GGC GTT AAT GGG GCG Ser Thr Tyr Leu lie Asp Asp Val Leu Tyr Thr Gly Val Asn Gly Ala 290 295 300 912 GTA TAT ACA TTT TCA AAT AAT GAA CTA AAC AAA ACT GGT TTA ACT AAT Val Tyr Thr Phe Ser Asn Asn Glu Leu Asn Lys Thr Gly Leu Thr Asn 305 310 315 320 960 AAC AAT AAT TAT ATC ACA ACA TCT ATA AAA GTA GAG GAT ACA TTA GTA Asn Asn Asn Tyr lie Thr Thr Ser lie Lys Val Glu Asp Thr Leu Val 325 330 335 1008 TGC GGA ACC AAT AAC GGA AAC CCC AAA TGT TGG AAA ATA GAC GGT TCC Cys Gly Thr Asn Asn Gly Asn Pro Lys Cys Trp Lys lie Asp Gly Ser 340 345 350 1056 GAA GAT CCA AAA TAT AGA GGT AGA GGA TAT GCT CCT TAT CAA AAT AGT Glu Asp Pro Lys Tyr Arg Gly Arg Gly Tyr Ala Pro Tyr Gin Asn Ser 355 360 365 1104 AAA GTG ACG ATA ATC AGT CAT AAC GAA TGT GTA CTA TCT GAT ATA AAC Lys Val Thr lie lie Ser His Asn Glu Cys Val Leu Ser Asp lie Asn 370 375 380 1152 ATA TCA AAA GAA GGA ATT AAA AGA TGG AGA AGA TTT GAC GGA CCA TGT lie Ser Lys Glu Gly lie Lys Arg Trp Arg Arg Phe Asp Gly Pro Cys 385 390 395 400 1200 17 Printed from Mimosa WO 99/21997 PCT/US98/22879 GGT TAT GAT TTA TAC ACG GCA GAT AAC GTG ATT CCA AAA GAT GGT GTG Gly Tyr Asp Leu Tyr Thr Ala Asp Asn Val lie Pro Lys Asp Gly Val 405 410 415 1248 CGT GGA GCA TTC GTT GAT AAA GAC GGC ACT TAT GAC AAA GTT TAC ATT Arg Gly Ala Phe Val Asp Lys Asp Gly Thr Tyr Asp Lys Val Tyr lie 420 425 430 1296 CTT TTC ACT GAT ACT ATC GAC ACA AAG AGA ATT GTT AAA ATT CCG TAT Leu Phe Thr Asp Thr lie Asp Thr Lys Arg lie Val Lys lie Pro Tyr 435 440 445 1344 ATA GCA CAA ATG TGC TTA AAT GAC GAA GGT GGT CCA TCA TCA TTG TCT lie Ala Gin Met Cys Leu Asn Asp Glu Gly Gly Pro Ser Ser Leu Ser 450 455 460 1392 AGT CAT AGA TGG TCG ACG TTT CTC AAG GTC GAA TTA GAA TGT GAT ATC Ser His Arg Trp Ser Thr Phe Leu Lys Val Glu Leu Glu Cys Asp lie 465 470 475 480 1440 GAC GGA AGA AGT TAT AGA CAA ATT ATT CAT TCT AAA GCT ATA AAA ACA Asp Gly Arg Ser Tyr Arg Gin lie lie His Ser Lys Ala lie Lys Thr 485 490 495 1488 GAT AAT GAT ACG ATA CTA TAT GTA TTC TTT GAT AGT CCT TAT TCC AAG Asp Asn Asp Thr lie Leu Tyr Val Phe Phe Asp Ser Pro Tyr Ser Lys 500 505 510 1536 TCC GCA TTA TGT ACC TAT TCT ATG AAT GCC ATT AAA CAC TCT TTT TCT Ser Ala Leu Cys Thr Tyr Ser Met Asn Ala lie Lys His Ser Phe Ser 515 520 525 1584 ACG TCA AAA TTG GGA GGA TAT ACA AAG CAA TTG CCG TCT CCA GCT CCT Thr Ser Lys Leu Gly Gly Tyr Thr Lys Gin Leu Pro Ser Pro Ala Pro 530 535 540 1632 GGT ATA TGT CTA CCA GCT GGA AAA GTT GTT CCA CAT ACC ACG TTT GAC Gly lie Cys Leu Pro Ala Gly Lys Val Val Pro His Thr Thr Phe Asp 545 550 555 560 1680 ATC ATA GAA CAA TAT AAT GAG CTA GAT GAT ATT ATA AAG CCT TTA TCT lie lie Glu Gin Tyr Asn Glu Leu Asp Asp lie lie Lys Pro Leu Ser 565 570 575 1728 CAA CCT ATC TTC GAA GGA CCG TCT GGT GTT AAA TGG TTC GAT ATA AAG Gin Pro lie Phe Glu Gly Pro Ser Gly Val Lys Trp Phe Asp lie Lys 580 585 590 1776 GAG AAG GAA AAT GAA CAT CGG GAA TAT AGA ATA TAC TTC ATA AAA GAA Glu Lys Glu Asn Glu His Arg Glu Tyr Arg lie Tyr Phe lie Lys Glu 595 600 605 1824 AAT ACT ATA TAT TCG TTC GAT ACA AAA TCT AAA CAA ACT CGT AGT GCA Asn Thr lie Tyr Ser Phe Asp Thr Lys Ser Lys Gin Thr Arg Ser Ala 610 615 620 1872 18 Printed from Mimosa -WO 99/21997 PCT/US98/22879 CAA GTT GAT GCG CGA CTA TTT TCA GTA ATG GTA ACT TCG AAA CCG TTA 1920 Gin Val Asp Ala Arg Leu Phe Ser Val Met Val Thr Ser Lys Pro Leu 625 630 635 640 TTT ATA GCA GAT ATA GGG ATA GGA GTA GGA ATA CCA CGA ATG AAA AAA 1968 Phe lie Ala Asp lie Gly lie Gly Val Gly lie Pro Arg Met Lys Lys 645 650 655 ATA CTT AAA ATG TAA 1983 lie Leu Lys Met *;660;(2) INFORMATION FOR SEQ ID NO:8;(l) SEQUENCE CHARACTERISTICS;(A) LENGTH 661 amino acids;(B) TYPE, ammo acid (D) TOPOLOGY linear;(n) MOLECULE TYPE protein;(xi) SEQUENCE DESCRIPTION SEQ ID NO 8;Met Phe His Val Ser Phe Arg Tyr lie Phe Gly lie Pro Pro Leu lie 15 10 15;Leu Val Leu Leu Pro Val Thr Ser Ser Asp Tyr Lys Asp Asp Asp Asp 20 25 30;Lys Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 35 40 45;Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 50 55 60;Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 65 70 75 80;Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 85 90 95;Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 100 105 110;Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 115 120 125;Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 130 135 140;Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 145 150 155 160;Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 165 170 175;19;Printed from Mimosa;-WO 99/21997 PCT/US98/22879;Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 180 185 190;lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 195 200 205;Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 210 215 220;Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 225 230 235 240;Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 245 250 255;Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 260 265 270;Ser Thr Ser lie Glu Trp His Lys Phe Glu Thr Ser Glu Glu lie lie 275 280 285;Ser Thr Tyr Leu lie Asp Asp Val Leu Tyr Thr Gly Val Asn Gly Ala 290 295 300;Val Tyr Thr Phe Ser Asn Asn Glu Leu Asn Lys Thr Gly Leu Thr Asn 305 310 315 320;Asn Asn Asn Tyr He Thr Thr Ser lie Lys Val Glu Asp Thr Leu Val 325 330 335;Cys Gly Thr Asn Asn Gly Asn Pro Lys Cys Trp Lys lie Asp Gly Ser 340 345 350;Glu Asp Pro Lys Tyr Arg Gly Arg Gly Tyr Ala Pro Tyr Gin Asn Ser 355 360 365;Lys Val Thr lie lie Ser His Asn Glu Cys Val Leu Ser Asp lie Asn 370 375 380;lie Ser Lys Glu Gly lie Lys Arg Trp Arg Arg Phe Asp Gly Pro Cys 385 390 395 400;Gly Tyr Asp Leu Tyr Thr Ala Asp Asn Val lie Pro Lys Asp Gly Val 405 410 415;Arg Gly Ala Phe Val Asp Lys Asp Gly Thr Tyr Asp Lys Val Tyr lie 420 425 430;Leu Phe Thr Asp Thr lie Asp Thr Lys Arg lie Val Lys lie Pro Tyr 435 440 445;lie Ala Gin Met Cys Leu Asn Asp Glu Gly Gly Pro Ser Ser Leu Ser 450 455 460;Ser His Arg Trp Ser Thr Phe Leu Lys Val Glu Leu Glu Cys Asp lie 465 470 475 480;20;Printed from Mimosa;WO 99/21997 PCT/US98/22879;Asp Gly Arg Ser Tyr Arg Gin lie lie His Ser Lys Ala lie Lys Thr 485 490 495;Asp Asn Asp Thr lie Leu Tyr Val Phe Phe Asp Ser Pro Tyr Ser Lys 500 505 510;Ser Ala Leu Cys Thr Tyr Ser Met Asn Ala lie Lys His Ser Phe Ser 515 520 525;Thr Ser Lys Leu Gly Gly Tyr Thr Lys Gin Leu Pro Ser Pro Ala Pro 530 535 540;Gly lie Cys Leu Pro Ala Gly Lys Val Val Pro His Thr Thr Phe Asp 545 550 555 560;lie lie Glu Gin Tyr Asn Glu Leu Asp Asp lie lie Lys Pro Leu Ser 565 570 575;Gin Pro lie Phe Glu Gly Pro Ser Gly Val Lys Trp Phe Asp lie Lys 580 585 590;Glu Lys Glu Asn Glu His Arg Glu Tyr Arg lie Tyr Phe lie Lys Glu 595 600 605;Asn Thr lie Tyr Ser Phe Asp Thr Lys Ser Lys Gin Thr Arg Ser Ala 610 615 620;Gin Val Asp Ala Arg Leu Phe Ser Val Met Val Thr Ser Lys Pro Leu 625 630 635 640;Phe lie Ala Asp lie Gly lie Gly Val Gly lie Pro Arg Met Lys Lys 645 650 655;lie Leu Lys Met * 660 (2) INFORMATION FOR SEQ ID NO 9 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 33 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS. single (D) TOPOLOGY linear (ll) MOLECULE TYPE primer (ill) HYPOTHETICAL NO (IV) ANTI-SENSE NO (XI) SEQUENCE DESCRIPTION' SEQ ID NO" 9-ATCGCATCAT CTACCTTCAT CCATTCCGAC CTG 33 (2) INFORMATION FOR SEQ ID NO'10 21 Printed from Mimosa WO 99/21997 PCT/US98/22879 (x) SEQUENCE CHARACTERISTICS (A) LENGTH 3 3 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS' single (D) TOPOLOGY linear (11) MOLECULE TYPE primer (ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO (xi) SEQUENCE DESCRIPTION SEQ ID NO 10. TAAACACTCC GAACAGGATT TATGTTTATT GCA 33 22 Printed from Mimosa