AU748168B2 - Viral encoded semaphorin protein receptor DNA and polypeptides - Google Patents

Description

SWO 99/21997 PCTIUS98/22879 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 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 semaphorin II are selectively involved in repulsive growth cone guidance during development. Flies having semaphorin II genes that are mutated so that their function is reduced exhibit abnormal behavior characteristics.

Another semaphorin gene has been identified in several strains of poxvirus.

This semaphorin is found in vaccinia virus (Copenhagen strain) and is encoded in an open reading frame (ORF) known as A39R. The A39R encoded protein has no transmembrane domain and no potential membrane linkage and is known to be a secreted protein. A variola 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 semaphorin, AHV-sema, has been found in the Alcelaphine Herpesvirus

(AHV).

Genes encoding mammalian (human, rat, and mouse) semaphorins have been identified, based upon their similarity to insect semaphorins. Functional studies of these semaphorins suggest that embryonic and adult neurons require a semaphorin to establish workable connections. Significantly, the fast response time of growth cone cultures to appropriate semaphorins suggests that semaphorin signaling involves a receptor-mediated signal transduction mechanism. To date, one semaphorin receptor, designated neuropilin, has been isolated using mRNA from rat spinal cord. Another -:WO 99/21997 PCT/US98/22879 receptor, designated neuropilin-2, has been suggested (Kolodkin et al. Cell 90:753- 762, 1997) Semaphorin ligands that are secreted into the extracellular milieu signal through receptor bearing cells in a local and systemic fashion. In order to further investigate the nature of cellular processes regulated by such local and systemic signaling, it would be beneficial to identify additional semaphorin receptors and ligands. Furthermore, because virus encoded semaphorins 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 primary function is to modify neurologic responses. It is more likely that the virus encoded semaphorins function to modify the immunologic response of the infected host and it is likely that mammalian homologues to virus encoded semaphorins function to modify the immunologic response. In view of the suggestion that viral semaphorins may function in the immune system as natural immunoregulators it would be beneficial to identify semaphorin receptors as therapeutic agents for enhancing or downregulating the immune response.

SUMMARY OF THE INVENTION The present invention pertains to semaphorin receptors as isolated or homogeneous proteins. In particular, the present invention provides a semaphorin receptor polypeptide, designated VESPR (Viral Encoded Semaphorin Protein Receptor) that binds semaphorins, including, but not limited to, the A39R vaccinia semaphorin and AHV semaphorin. Also, within the scope of the present invention are DNAs encoding VESPR polypeptides and expression vectors that include DNA encoding VESPR polypeptides. The present invention also includes host cells that have been transfected or transformed with expression vectors that include DNA encoding a VESPR polypeptide, and processes for producing VESPR polypeptides by culturing 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 purifying or separating semaphorins or cells that express semaphorins to which the VESPR polypeptides of the present invention bind. Such processes include binding at least one VESPR polypeptide to a solid phase matrix and contacting a mixture containing a semaphorin polypeptide to which the VESPR polypeptide binds, or a mixture of cells expressing the semaphorin with the bound VESPR polypeptide, and then separating the contacting surface and the solution.

3 The present invention additionally provides processes for treating inflammation and inflammatory disease. Such processes include administering a therapeutically effective amount of a soluble VESPR polypeptide to a human or other mammal afflicted with disease associated with proinflammatory activity of a semaphorin ligand.

The present invention provides an isolated VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, the binding portion being capable of binding a semaphorin.

The present invention further provides an isolated VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide encoded by DNA selected from the group consisting of: DNA of SEQ ID NO:1; S: DNA sequences that hybridize under moderately stringent conditions to S the DNA of and which DNA sequences encode a polypeptide that binds o* semaphorins; and 0 DNA complementary to the DNA of and Another aspect of the present invention is a soluble VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: amino acids xl to 944 of SEQ ID NO:2, wherein x, is amino acid 1 or 35; and a fragment of the sequence of wherein the binding portion is capable of binding a semaphorin.

3a A further aspect of the invention is a method of screening for binding to a VESPR 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 of: SEQ ID NO:2; an amino acid sequence that is at least 90% identical to SEQ ID NO:2; an amino acid sequence that is at least 90% identical to amino acids xl to 944 of SEQ ID NO:2 wherein x, is amino acid 1 or 35; and fragments of or wherein the binding portion is capable of binding a semaphorin.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel semaphorin receptor polypeptides designated Viral Encoded Semaphorin Protein Preceptor (VESPR), DNA encoding VESPR polypeptides and recombinant expression vectors that include 20 DNA encoding VESPR polypeptides. The present invention additionally provides methods for isolating VESPR polypeptides and methods for producing 'i recombinant VESPR polypeptides by cultivating host cells transfected with the recombinant expression vectors under conditions appropriate for expressing semaphorin receptors and recovering the expressed receptor polypeptide.

2 In particular, the present invention provides VESPR polypeptides that bind semaphorins, including but not limited to, the vaccinia virus A39R semaphorin and the AHV semaphorin. The native VESPR polypeptide described herein was isolated using an Ectromeliavirus A39R semaphorin (Fc fusion protein (A39R/Fc) to recover VESPR from the membranes of human cells expressing the receptor. As described in the examples below, flow cytometry experiments establish that the VESPR polypeptides of the present invention are expressed by B cells lines, monocyte-type cell lines, T cell lines, dendritic cells, NK cells, lung epithelial cells, stroma, intestinal epithelial cells and lymphoma cells.

W:llona/Sharon/speci/sp12047 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 dendritic cells. Also characteristic of semaphorin receptors described herein is their ability to interact with their ligands to synergise with interferon and SAC to upregulate IL-12 production and down regulate MHC class II and CD86 expression on mouse dendritic 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 semaphorins and their receptors. Among the uses of the VESPR polypeptides that flow from aforementioned biological properties of the receptor-ligand interaction are inducing IL-12 production and subsequent natural killer cell activation. VESPR polypeptides find further use in treating diseases and adverse conditions associated with i inflammation. In particular, soluble VE3PR 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 al., Biochem and Biophys Res Corn, 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 S 15 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.

S. 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 described 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 25 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 amino acids 966-1568.

•A DNA encoding amino acids 19-1100 of human VESPR in E. coli DH 10B was deposited with the American type Culture Collection, Rockville, MD, USA on 22 October 3 1997 and assigned accession number ATCC 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: 1 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 and DNA sequences found in data bases obtained from the National Center for Biotechnology Information. Similarity scores obtained as a result S WO 99/21997 PCT/US98/22879 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" (Maestrini et al., 1996, and Kameyama et al., 1996). Pairwise and multiple sequence alignments between VESPR and human and murine members of the plexin family were performed using the Smith-Waterman algorithm as implemented in the Genetics Computer Group (GCG) programs "BESTFIT" and "PILEUP" (Wisconsin Package, 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 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.

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 subdomain with similarity to a region of the semaphorin domain of a number of members of the semaphorin family of genes described 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, forming a consensus sequence of where x is any amino acid. This entire subdomain is distinct from the canonical semaphorin domain described for the semaphorin gene family in that it is smaller (100 amino acid residues for the subdomain vs 500 residues for the entire semaphorin domain), it is also present in the plexin gene family and MET-hepatocyte growth factor receptor family, neither of which is a canonical semaphorin gene family members, and it is present in a gene which is not itself a member of the semaphorin gene family but which interacts with a member of the semaphorin family (A39R). These subdomain sequences, therefore, represent peptides that are potentially capable of further identifying other receptors which interact with semaphorins.

-WO 99/21997 PCT/US98/2879 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 in SEQ ID NO:1. As described in Example 5, identifying the cDNA that encodes the amino acid sequence of SEQ ID NO:2 enables constructing expression vectors that include the encoding cDNAs. Then culturing host cells transfected with a recombinant expression vector that contains cDNA encoding VESPR polypeptide, under conditions appropriate for expressing the VESPR polypeptide, and recovering the expressed VESPR polypeptide provides methods for producing VESPR polypeptides of the present invention.

Since VESPR polypeptide is found in B cell lines, T cell lines and dendritic cells, treating a variety 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 described infra.

The terms "VESPR" and "VESPR polypeptide" of the present invention 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 similarity or a high degree of identity with the amino acid sequence of SEQ ID NO:2, which polypeptides are biologically active and bind at least one molecule or fragments of a molecule that are members of the semaphorin family. In addition, the term VESPR refers to biologically active gene products of the DNA of SEQ ID NO: 1.

Further encompassed by the term VESPR are soluble or truncated proteins that comprise primarily the binding portion of the protein, retain biological activity and are capable of being secreted. Specific examples of such soluble proteins are those comprising the sequence of amino acids 1-944 of SEQ ID NO:2.

The term "biologically active" as it refers to VESPR or semaphorin receptor polypeptide, means that the VESPR or semaphorin receptor polypeptide is capable of binding to at least one semaphorin. Assays suitable for determining VESPR binding are described herein and can include standard flow cytometry tests and slide binding tests.

"Isolated" means a VESPR is substantially free of association with other proteins or polypeptides residual of the expression process, for example, as a purification product of recombinant host cell culture or as a purified extract.

A VESPR variant as referred to herein, means a polypeptide substantially homologous to native VESPR, but which has an amino acid sequence different from WO 99/21997 PCT/US98/22879 that of native VESPR because of one or more deletions, insertions or substitutions.

The variant 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 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.

Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, 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 characteristics, are well known. Naturally occurring VESPR variants or alleles are also encompassed by the invention. Examples of such variants 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 occurring soluble form of the protein, for example.

Variations attributable to proteolysis include, for example, differences in the Ntermini 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 terminal amino acids).

As mentioned above, Example 1 describes the construction of novel viral A39R/Fc fusion proteins useful in studying VESPR binding. Other antibody Fc regions may be substituted for the human IgG1 Fc region described 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 IgG1 or fragments of the human or murine IgG1 Fc region, fragments comprising at least the hinge region so that interchain disulfide bonds will form. The viral A39R:Fc fusion protein offers the advantage of WO 99/21997 PCTIS98/22879 being easily purified. In addition, disulfide bonds form between the Fc regions of two separate fusion protein chains, creating dimers.

As described above, in one aspect, the present invention includes soluble VESPR polypeptides. Soluble VESPR polypeptides comprise all or part of the extracellular domain of a native VESPR but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble VESPR polypeptides advantageously comprise 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 semaphorin ligands. Indeed, 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 protein from the culture medium, by centrifugation, 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 native, membrane bound VESPR protein. Purification of the proteins from recombinant host cells is feasible, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration.

Examples of soluble VESPR polypeptides include those comprising a 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 comprising less than the entire extracellular domain are included in the invention, e.g. amino acids 35-944. When initially expressed within a host cell, soluble VESPR polypeptides may additionally comprise one of the heterologous signal peptides described below that is functional within the host cells employed. Alternatively, the protein may comprise the native signal peptide. In one embodiment of the invention, soluble VESPR can be expressed as a fusion protein comprising (from N- to C-terminus) the yeast a-factor signal peptide, a FLAG® peptide described below and in U.S. Patent No. 5,011,912, and soluble VESPR polypeptide consisting of amino acids 1-944 or 35-944 of SEQ ID C, WO 99/21997 PCTfUS98/22879 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 enterokinase. Isolated DNA sequences encoding soluble VESPR proteins are encompassed by the invention.

Truncated VESPR polypeptides, including soluble polypeptides, may be 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 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, immediately downstream of the codon for the last amino acid of the binding domain.

As stated above, the invention provides isolated or homogeneous VESPR polypeptides, both recombinant and non-recombinant. Variants and derivatives of native VESPR proteins that retain the desired biological activity the ability to bind to semaphorins) may be obtained by mutations of nucleotide sequences coding 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 particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog 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 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. 154:367, 1987); and U.S. Patent Nos. 4,518,584 and 4,737,462 all of which are incorporated by reference.

WO 99/21997 PCT/US98/22879 Native VESPR polypeptide may be modified to create VESPR derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives 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 VESPR polypeptide or the extracellular domain thereof. Other derivatives 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-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence 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 comprise peptides added to facilitate purification and identification of VESPR. Such peptides include, for example, poly- His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.

The invention further includes VESPR with or without associated nativepattern glycosylation. VESPR polypeptide expressed in yeast or mammalian expression systems 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 bacterial expression systems, such as E. coli, provides non-glycosylated molecules.

Equivalent DNA constructs that encode various additions or substitutions of 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 in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. The native human VESPR protein comprises 24 such triplets, 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, 895-897 and 920-922 of SEQ ID NO:2. Appropriate substitutions, additions or WO 99/21997 PCTIUS98/22879 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 Nglycosylation sites in proteins include those described in U.S. Patent 5,071,972 and 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 bridges upon renaturation. Other equivalents are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in 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 adjacent basic residues. Lys-Lys pairings 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 DNA and RNA sequences that hybridize to the VESPR nucleotide sequences disclosed herein under conditions of moderate or high stringency, and that encode biologically active VESPR. Conditions of moderate stringency, as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 101- 104, Cold Spring Harbor Laboratory Press, (1989), include use of a prewashing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55 0 C, 5 X SSC, overnight. Conditions of high stringency include higher temperatures of hybridization 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 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 variant DNA sequences may result from silent WO 99/21997 PCTIUS98/22879 mutations occurring during 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: cDNA comprising the nucleotide sequence presented in SEQ ID NO: 1; DNA capable of hybridization to a DNA of under moderately stringent conditions and that encodes biologically active VESPR polypeptide; DNA that is degenerate as a result of the genetic code to a DNA defined in or and that encodes biologically active VESPR polypeptide; and (d) DNA complementary to the DNA of or VESPR polypeptides encoded by such DNA equivalent sequences are encompassed by the invention.

DNAs that are equivalents to the DNA sequence of SEQ ID NO:1 will hybridize under moderately and highly stringent 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 comprising inactivated N-glycosylation site(s), inactivated KEX2 protease processing site(s), or conservative amino acid substitution(s), as described above. VESPR polypeptides encoded by DNA derived from other species, wherein the DNA will hybridize to the cDNA of SEQ ID NO:1 are also encompassed.

Variants possessing the requisite ability to bind semaphorins may be identified by any suitable assay. Biological activity of VESPR polypeptides may be determined, for example, by competition for binding to the receptor binding domain of semaphorins competitive binding assays).

One type of a competitive binding assay for a VESPR polypeptide uses a radiolabeled, soluble VESPR and intact semaphorin-expressing cells. Instead of intact cells, one could substitute soluble semaphorin:Fc fusion proteins bound to a solid phase through the interaction of a Protein A, Protein G or an antibody to the semaphorin or Fc portions of the molecule, with the Fc region of the fusion protein.

Another type of competitive binding assay utilizes radiolabeled soluble semaphorins such as a fusion protein, and intact cells expressing VESPR.

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 semaphorin ligand.

For example, a radiolabeled soluble semaphorin ligand can be antagonized by soluble VESPR in an assay for binding activity against a surface-bound semaphorin receptor.

-WO 99/21997 PCT/US98/22879 Qualitative results can be obtained by competitive autoradiographic plate binding assays, or Scatchard plots may be utilized to generate quantitative results.

Alternatively, semaphorin binding proteins, such as VESPR or antisemaphorin antibodies, can be bound to a solid phase such as a column chromatography matrix or a similar substrate suitable for identifying, separating or purifying cells that express semaphorin on their surface. Binding of a semaphorinbinding 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. Various other means for fixing proteins to a solid phase are well known in the art and are suitable for use in 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 semaphorin-expressing cells are contacted with the solid phase that has VESPR polypeptides thereon. Cells having semaphorin on their surface bind to the fixed VESPR and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening or separating such semaphorin-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-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner. In the case of semaphorin-VESPR interactions, the enzyme preferably would cleave the semaphorin, thereby freeing the resulting cell suspension from the "foreign" semaphorin receptor material. The purified cell population then may be used to repopulate mature (adult) tissues.

Alternatively, mixtures of cells suspected of containing semaphorin-positive cells first can be incubated with biotinylated VESPR. Incubation periods are typically at least one hour in duration to ensure sufficient binding to semaphorin The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin 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., 10D:239 (1986). Washing unbound material and releasing the bound cells is performed using conventional methods.

As described above, VESPR can be used to separate cells expressing semaphorin. In an alternative method, VESPR or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as 1251 to detect semaphorin-expressing cells. Radiolabeling with 125I can be performed by any of WO 99/21997 PCT/US98/22879 several standard methodologies that yield a functional 1 25 I-VESPR molecule labeled to high specific activity. Or an iodinated or biotinylated antibody against the semaphorin receptor can be used. Another detectable moiety such as an enzyme that can catalyze a colorimetric or fluorometric reaction, biotin or avidin may be used.

Cells to be tested for semaphorin-expression can be contacted with labeled VESPR polypeptide. After incubation, unbound labeled VESPR is removed and binding is measured using the detectable moiety.

The binding characteristics of VESPR (including variants) may also be determined using a conjugated semaphorin (for example, 1 2 5 I-semaphorin:Fc) in competition assays similar to those described above. In this case, however, intact cells expressing semaphorins bound to a solid substrate are used to measure the extent to which a sample containing a putative VESPR variant competes for binding with a conjugated semaphorin.

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 that express semaphorin and proliferate in the presence of VESPR.

The VESPR proteins disclosed herein also may be employed to measure the biological activity of semaphorin 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 semaphorin protein chemical modification, truncation, mutation, etc.). The biological activity of a semaphorin 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, to monitor shelf life and stability of semaphorin protein under different conditions. To illustrate, VESPR polypeptides may be employed in a binding affinity study to measure the biological activity of an semaphorin protein that has been stored at different temperatures, or produced in different cell types. The binding affinity of the modified semaphorin protein for VESPR is compared to that of an unmodified semaphorin protein to detect any adverse impact of the modifications on biological activity of the semaphorin.

VESPR polypeptides also find use as carriers for delivering agents attached thereto to cells expressing semaphorins. As described in example 7 below, a putative human semaphorin is expressed in cells found in the placenta, testis, ovary and spleen. VESPR polypeptides can thus can be used to deliver diagnostic or therapeutic -WO 99/21997 PCT/US98/22879 agents to these cells (or to other cell types found to express a semaphorin 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 colorimetric or fluorometric reaction, and the 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-phenylalanine nitrogen mustard or cyclophosphamide, intercalating agents such as cis-diaminodichloroplatinum, antimetabolites such as fluorouracil, vinca alkaloids such as vincristine, and antibiotics such as bleomycin, doxorubicin, daunorubicin, and derivatives thereof. Among the toxins are ricin, abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, 1231, 1311, 9 9 mTc, 11lIn, and 76 Br. Radionuclides suitable for therapeutic use include, but are not limited to, 1311, 2 1 1 At, 7 7Br, 18 6 Re, 1 8 8 Re, 2 12 pb, 2 12 Bi, 10 9 pd, 64 Cu, and 6 7 Cu.

Such agents may be attached to the semaphorin receptor by any suitable conventional procedure. VESPR, being a protein, comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the protein or agent may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling proteins are known.

Radionuclide metals may be attached to the receptor by using a suitable bifunctional chelating agent, for example.

Conjugates comprising VESPR and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application.

Another use of the VESPR of the present invention is as a research tool for studying the role that the receptor, in conjunction with semaphorins, 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 semaphorin to which it binds or VESPR, or the interactions thereof.

S:WO 99/21997 PCT/US98/22879 As described in Example 16 semaphorins 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 dendritic cells. The use of VESPR and its semaphorin ligand to induce IL-12 production promotes natural killer cell and T cell production and induces cytokine production (primarily y-interferon).

IL-12 and 1L-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 promote production of cytokines associated with Th2 cell differentiation, thus discouraging proinflammatory activity. Also, VESPR in combination with its semaphorin 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 a more persistent Thl-type immunological memory.

Furthermore, it is known that administration of IL-12 to tumor bearing animals results in tumor regression and the establishment of a tumor-specific immune response. Thus, using a semaphorin ligand to bind with VESPR in order to enhance or promote IL-12 can induce a curative immune response against aggressive micrometastasizing tumors.

Additionally, as described in example 18, receptors of the present invention bind with their semaphorin ligands to increase CD54 expression on monocytes. This observation suggests that the semaphorin/semaphorin receptor interaction mediate cellular activation that contributes to the proinflammatory activity typically associated with monocyte activation. Such activity includes increased phagocytosis, pinocytosis, nitric oxide production and cytokine production. To antagonize or reverse the proinflammatory activity resulting from the interaction between the semaphorin ligand and its membrane bound receptor, a pharmaceutical composition containing a therapeutically effect amount of a soluble VESPR of the present invention can be administered parenterally to an organism. The soluble VESPR binds with the semaphorin ligand thus preventing the ligand from binding with a membrane bound receptor and contributing to the proinflammatory activity. A therapeutically effect amount of VESPR is an amount sufficient to antagonize proinflammatory activity.

: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 occurs during 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, 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 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.

Semaphorin ligands binding with VESPR to downregulate expression of MHC Class II molecules and CD86, a co-stimulatory molecule, on dendritic cells, cultured with GM-CSF and IL-4 (see example 17) suggests that the interaction between semaphorin ligands and the receptors of the present invention are associated with the immune suppression of mature dendritic cells. To antagonize or reverse the immunosuppression activity resulting from the interaction between the semaphorin ligand and its membrane bound receptor, a pharmaceutical composition containing a therapeutically effective amount of a soluble VESPR of the present invention can be administered parenterally to an organism. The soluble VESPR binds with the semaphorin ligand thus preventing the ligand from binding with a membrane bound receptor and contributing to the immunosuppression activity. Alternatively, in patients or organisms that suffer from the effects of chronic inflammation, administering appropriate semaphorin ligands will contribute 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 dendritic cells from the T cell zones of lymph nodes.

Additionally, data suggests that VESPR is involved in shutting down the T cell immune response once a pathogen has been cleared.

-WO 99/21997 PCTIUS98/22879 VESPR polypeptides of the invention can be formulated according to known methods used to prepare pharmaceutically useful compositions. VESPR can be combined in admixture, either as the sole active material or with other known active materials, with pharmaceutically suitable diluents Tris-HC1, acetate, phosphate), preservatives Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable carriers and their formulations are described in 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 into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of VESPR. VESPR polypeptide can also be conjugated to antibodies against tissuespecific receptors, ligands or antigens, or coupled to ligands of tissue-specific receptors.

VESPR polypeptides can be administered topically, parenterally, or by inhalation. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. These compositions will typically contain an effective amount of the VESPR, alone or in combination with an effective amount of any other active material. 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 practices.

VESPR polypeptides may exist as oligomers, such as covalently-linked or non-covalently-linked dimers or trimers. 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 region of an antibody IgG1) in a manner that does not interfere with binding of VESPR to a semaphorin ligand-binding domain. The Fc polypeptide preferably is fused to the C-terminus of a soluble VESPR (comprising only the ligand-binding domain). General preparation of fusion proteins comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (PNAS USA 88:10535, WO 99/21997 PCT/US98/22879 1991) and Byrn et al. (Nature 344:677, 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 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 with bacterial, fungal, yeast, and mammalian cellular hosts are described, 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, 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 prokaryotic host cell. The N-terminal methionine may be cleaved from the expressed recombinant VESPR polypeptide.

VESPR polypeptides may be expressed in yeast host cells, preferably from the Saccharomvces genus S. cerevisiae). Other genera of yeast, such as Pichia K.

lactis or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2j 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 metallothionein, 3phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 :WO 99/21997 PCTIUS98/22879 or in Fleer 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 described by Russell et al. Biol. Chem. 258:2674, 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 replication in E. coli (Ampr gene and origin of replication) into the above-described 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, Kurjan et al., Cell 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 restriction sites. This will facilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art. One such protocol is described 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, casamino acids, 2% glucose, 10 lg/ml adenine and 20 pig/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 [ig/ml adenine and 80 lg/ml uracil. Depression of the ADH2 promoter occurs when glucose is exhausted from the medium.

Mammalian or insect host cell culture systems could also be employed to express recombinant VESPR polypeptides. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 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-1/EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).

: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 derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, 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 origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.

Exemplary expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A- 0367566, and in U.S. Patent Application Serial No. 07/701,415, filed May 16, 1991, incorporated by reference herein. The vectors may be derived 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 described in United States Patent 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL-4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II IL-1 receptor signal peptide described in EP 460,846.

VESPR polypeptides as isolated, purified or homogeneous proteins according to the invention may be produced by recombinant expression systems as described above or purified from naturally occurring cells. VESPR can be purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDSpolyacrylamide gel electrophoresis (SDS-PAGE).

One process for producing VESPR comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes VESPR polypeptide under conditions sufficient to promote expression of VESPR polypeptide. The receptor is then recovered from culture medium or cell extracts, :WO 99/21997 PCT/US98/2879 depending upon the expression system employed. As is known to the skilled artisan, procedures for purifying a recombinant 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 are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.

Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.

Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP- HPLC media, silica gel having pendant methyl or other aliphatic groups) can be employed to further purify VESPR polypeptide. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide a substantially homogeneous recombinant protein.

It is possible to utilize an affinity column comprising the receptor-binding domain of a semaphorin that binds VESPR to affinity-purify expressed VESPR polypeptides. VESPR polypeptides can be removed from an affinity column using conventional techniques, 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 affinity matrix utilized. Alternatively, the affinity column may comprise an antibody that binds VESPR. Example 20 describes a procedure for employing VESPR of the invention to generate monoclonal antibodies directed against VESPR Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freezethaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

WO 99/21997 PCT/US98/22879 Transformed yeast host cells are preferably employed to express VESPR as a secreted polypeptide in order to simplify purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. Chromatog. 296:171, 1984). Urdal et al.

describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.

Useful fragments of the VESPR nucleic acids include antisense or sense oligonucleotides comprising 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 invention, comprise a fragment of the coding region of VESPR cDNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of VESPR proteins.

Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo 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 described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid 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 the target nucleic acid sequence by any gene transfer method, including, for example, WO 99/21997 PCT/US98/2879 CaPO4-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 vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived 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 containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described 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 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 oligonucleotidelipid complex, as described in WO 90/10448. The sense or antisense oligonucleotidelipid 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 describes preparation of an Ectromelia Semaphorin A39R/immunoglobulin fusion protein (A39R/Fc). The process included preparing 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 fusion protein was used as described in Examples 3, 4, 5 and 6 to study VESPR binding characteristics and isolate VESPR.

DNA encoding A39R semaphorin was isolated and amplified from genomic Ectromelia virus DNA using PCR techniques and synthesized oligonucleotide primers whose sequences were based on published A39R sequences in the Copenhagen strain of Vaccinia Virus. The Copenhagen strain A39R DNA sequence is described in WO 99/21997 PCT/US98/22879 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 ATAAGTTTGA A (SEQ ID NO:3) Spe 1 A39R DNA Downstream Notl primer: GACAGCGGCC GCCTATTACA TTTTAAGTAT TTT (SEQ ID NO:4) Not 1 A39R DNA A Bgl II to Nsl I restriction fragment containing a mutein human Fc region of immunoglobulin as described by Baum et al. Cir. Sh. 44:30 (1994) was ligated into an expression vector (pDC304) containing a murine IL-7 signal peptide and a

FLAG

T M octapeptide as described 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 murine IL-7 signal peptide and FLAGTM peptide, in 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 pm filter. Then approximately 1 L of culture supernatant was passed through a BioCad Protein A HPLC protein purification 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 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 purified fusion protein was neutralized as it eluted using IM HEPES solution at pH 7.4.

-WO 99/21997 PCTIUS98/22879 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 harvesting supernatants 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 purification processes. After the Gly-Ser-6xHIS motif the primers added an in-frame termination codon and a Bgl 2 site. The PCR product was cut and cloned in pDC409 expression vector (McMahon et al., EMBO J. 10:2821,1991).

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 0.5% low immunoglobulin bovine serum, cell supernatants were harvested and a solution of 0.2% sodium azide was added to the supernatants. The supematants were filtered through a 0.22 ptm filter, concentrated 10 fold with a prep scale concentrator (Millipore; Bedford, MA) and purified on a BioCad HPLC protein purification equipped with a Nickel NTA Superflow self pack resin column (Qiagen, Santa Clarita, CA). After the supernatant passed through the column, the column was washed with Buffer A (20mM NaPO4, 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, concentrated 2 fold, and then dialyzed in PBS. The resulting A39R/polyHis fusion protein was then filtered through a 0.22pm sterile filter.

EXAMPLE 3 Screening Cell Lines for Binding to A39R The A39R/Fc fusion protein prepared as described 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 blocked cells were incubated with 5 pg/mL of A39R/Fc fusion protein in 2% FCS, -WO 99/21997 PCT/US98/22879 5% goat serum and 5% rabbit serum in PBS. Following the incubation the sample was washed 2 times with FACS buffer FCS in PBS) and then treated with mouse anti human Fc/biotin (purchased from Jackson Research) and SAPE (streptavidinphycoerythrin purchased from Molecular Probes). This treatment causes the antihuman Fc/biotin to bind to any bound A39R/Fc and the SAPE to bind to the antihuman 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 semaphorin 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 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 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 Peripheral 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 Derived Stroma) W126 (Human Lung Epithelium) PL-1 (Human T-Cell Clone VK-1 Human T-Cell Clone Primary Peripheral Blood T Cells Primary Human NK Cells RAJI (Burkitt's Lymphoma) KG1 (Human myeloid Cell Line) A39R Binding Result -WO 99/21997 PCT/US98/22879 THP-1 (Human Promonocytic Cell Line) MC6 (Mouse Mast Cell) EL4 (Mouse Thymoma) BeWo (Chorio Carcinoma) Primary Mouse Dendritic Cells Primary Human Dendritic 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 radiolabeling CB23 and human PB T cell surfaces, harvesting and treating cell supernatants 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 radiolabeling approximately 1x10 7 CB23 or PB T cells with [1251] as described by Benjamin et al.; Blood 75,:2017-2023 (1990). Cultured cell supernatants were harvested and clarified by centrifugation 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% Triton-X 100 containing protease inhibitors phenylmethylsulfonyl fluoride, Pepstatin-A, and Leupeptin. The lysates were clarified by centrifugation at 14,000 rpm for 30 minutes. In order to precipitate any receptor present in the lysate and/or supernatant, 200pL of the cell supernatant or lysate was incubated with 2pg of A39R/Fc fusion protein prepared as described in Example 1. The incubation was carried out for 1 hour with gentle rocking at 4 0 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 supernatants and the mixture was incubated for 1 hour with gentle rocking at 4°C. The beads were washed extensively with a PBS 1% Triton-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 semaphorin receptor was present in cell lysate and cell supernatant, confirming its expression as membrane bound protein and as a secreted soluble protein.

-WO 99/21997 PCT/US98/22879 Example Isolating and Sequencing a Semaphorin Receptor The A39R/Fc fusion protein, prepared as described in Example 1, was used to isolate a human semaphorin receptor polypeptide and a procedure for the isolated polypeptide purification was confirmed. The semaphorin receptor was isolated by suspending CB23 cell pellets in a solution of protease inhibitors that included 1 mM each of PMSF, Leupeptin, Aprotinin, Pepstatin A, 10g/mL APMSF, and 1 mM EDTA in homogenization buffer (10 mM phosphate, 30 mM NaCI, pH 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 0 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 Tris, 150 mM NaCI, the protease inhibitors identified above, 1% Triton X-100 and 0.1 mM of CaCI,, MgCl 2 and McCI, salts (Buffer The suspended pellets were dounced and spun in a SW-28 rotor for minutes at 25,000 rpm at 4'C. The supernatant was placed onto a 100mL wheat germ agglutinin column and allowed to elute at a rate of 1 mL/minute with 10 column volumes of Buffer A. Proteins 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 pIg of A39R/Fc fusion protein for 1 hour at 4 0 C. The incubated mixture was run through a sepharose column to remove material 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% Triton X-100 followed by a wash with PBS to wash off any unbound material.

Then proteins that were retained on the Protein A/Sepharose column were eluted in 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 uL using a 10 kD MWCO Centricon concentrator. Protein in the resulting concentrated sample were reduced and then alkylated using standard DTT and iodoacetic 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.

-WO 99/21997 PCTIUS98/22879 The approximately 200 kD band, located by comparison 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 dried slide. The slide was incubated at 37 0 C for 16 hours and then protein in the slice was extracted with acetonitrile with 5% formic acid three times while incubating 30 minutes with each extraction.

The trypsin digested peptide fragments were lyophilized, reconstituted in of 0.1% trifluoroacetic acid, and separated by RP-HPLC on a 500 .t id x 25 cm capillary column packed with C-18 reverse phase packing. The HPLC liquid phase was an acetonitrile/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-terminal 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 described above, were dried on a vacuum centrifuge and peptides in the fraction were dissolved in 6pL of 50% methanol containing 0.5% acetic acid. Two microliters (2 pL) of each of the peptide solutions were loaded into nanospray tips (Protein Analysis Company, Odense, Denmark).

Data were obtained with a Finnigan TSQ700 triple quadrupole mass spectrometer (San Jose, CA) equipped with a nanospray source. Mass spectra were acquired at unit 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 esterification was performed using standard esterification procedures.

The tandem mass spectrometry analysis of the trypsin generated peptides provided amino acid sequence information for isolated portions of the purified protein. The tandem mass spectral data were used in computer assisted screening of non-redundant protein databases and EST databases using the local SEQUEST algorithm search tool (Eng, J.K et al. J am Soc. Mass 1994). The peptide query sequences GluGluThrProValPheTyrLys corresponding to amino acids 421-428 of SEQ ID NO:2, and AsnIleTyrIleTyrLeuThrAlaGlyLys, 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 amino acids 388-401 identified EST No. R08946 as containing a 100% identity to the query.

:WO 99/21997 PCTIUS98/22879 The 100% identity between portions of EST 248534 and three peptide fragments of the purified protein strongly suggested that the cDNA contained within EST 248534 represented a portion of the nucleotide sequence for the coding region of the purified protein. A source of semaphorin receptor cDNA was identified using phage library screening methods and PCR primers based upon EST 248534.

The oligonucleotide primers had the following nucleotide sequences: ATCGCATCAT CTACCTTCAT CCATTCCGAC CTG (SEQ ID NO:9) TAAACACTCC GAACAGGATT TATGTTTATT GCA (SEQ ID NO: PCR isolation and amplification methodologies were carried out using a panel of human tissue cDNA phage libraries as templates for the PCR reactions. The PCR reaction mixture included 1pL of phage library stock, PCR oligonucleotide primers at 0.3 p.M final concentration, lx PC2 buffer (Ab Peptides, Inc., St. Louis, MO), 0.2 mM each of dATP, dCTP, dGTP, dTTP (Pharmacia Biotech) 0.2 pL of a 16:1 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 0 C for 5 minutes; thirty cycles at 98°C for 45 seconds, thirty cycles at 68 0 C for 45 seconds; thirty cycles at 72 0 C for seconds, and 1 cycle at 72 0 C for 5 minutes using a Robocycler 96 from Stratagene, La Jolla, CA. cDNAs in several libraries were positively identified as containing DNA encoding the purified VESPR protein based upon the appearance of an appropriate sized DNA band in electrophoresed PCR product.

Two of the phage libraries, human foreskin fibroblast and human dermal fibroblasts, were chosen for additional analysis. Libraries were plated according to established prodedures and probed with a radiolabeled random primer probe derived from a PCR amplification product using EST 248534 as a template. The PCR conditions used to obtained the amplification product were as described above and the probe was generated using Prime-IT II Random Primer Labeling Kit from Stratagene, La Jolla, CA Approximately 1 x 106 cpm/mL of purified probe was used to probe human foreskin phage libraries on nylon membrane filters overnight at 63 0 C in a hybridization buffer of 10x Denhardts solution, 50 mM Tris at pH 7.5, 0.9 M NaC1, 0.01% Sodium Pyrophosphate, 1% sodium dodecyl sulfate, and 200pg/mL denatured, fragmented salmon sperm DNA. After probing, the probed membranes were washed once in 6x SSC, 0.1% SDS for 20 minutes, once in 2 x SSC, 0.1% SDS for WO 99/21997 PCT/US98/22879 minutes, once in lx SSC, 0.1% SDS for 20 minutes, and once in 0.1 x SSC, 0.1% SDS for 20 minutes at 63 0 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 of VESPR as shown in SEQ ID NO: 1 and the amino 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 semaphorin. Purified A39R/Fc was prepared as described in Example 1 above. The purified protein was used to generate antibodies against A39R semaphorin as described in U.S. Patent 4,411,993. Briefly, mice were immunized at 0, 2 and 6 weeks with 10 pg with A39RIFc. The primary immunization was prepared with 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 pig A39R/Fc in PBS. Three days after the IV boost, splenocytes were harvested and fused with an Ag8.653 myeloma fusion partner using aqueous PEG 1500 solution. Hybridoma supernatants were screened for A39R 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 describes Northern Blot experiments carried out to identify 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 described 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 (EST 248534). A riboprobe template was generated using PCR techniques and oligonucleotide primers that were based on nucleotides 1-372 of EST 248534. The upstream and down stream primers that encompasses nucleotides 1-372 of EST 248534 had the following sequences: GCGGGACTCA GAGTCACC (SEQ ID :WO 99/21997 PCT/US98/22879 GGATCCTAAT ACGACTCACT ATAGGGAGGA AACCACTCCG AAC (SEQ ID NO:6) The underlined portion is a T7 site.

The two primers were used to isolate and amplify a PCR product from EST248534 for use in generating a riboprobe. The riboprobe was generated using Ambion's MAXIscript SP6/T7 kit by combining 3pL of RNAse free water, 2pL transcription buffer, 1pL each of 10mM dATP/dCTP/dGTP, 5.tL EST 248534 PCR Product, 5 iL Amersham [ac 2 P]UTP 10mCi/mL, 2pL T7 RNA polymerase at room temperature. The combination was microfuged, spun briefly, and incubated at 37 0 C for 30 minutes. Then 1pL DNAse was added to the mixture and allowed to react for 15 minutes at 37 0 C. The reaction product was passed through two column volumes of G-25 packing (Boehringer). One microliter (lCL) of the riboprobe was counted in a scintillation counter for 1 minute to determine cpm/mL Northern blots were generated by fractionating polyadenylated RNA from a variety 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 described 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., San Leandro, CA Cat #s 021001, 021002, 021003).

The Northern blots were prehybridized in a 50% formamide hybridization solution (30 mL 20x SSC, 2 mL 100x Denhardt's reagent, 1 mL of 10 mg/mL denatured fragmented salmon sperm DNA, 50 mL 100% formamide and 20 mL SDS. The total RNA blots were pre-hybridized at 42 0 C for 4 hours and the polyA+ RNA blots were pre-hybridized at 63 0 C for 4 hours. The riboprobe was added to clean hybridization solution (same as prehybridization solution) at a count of 10 6 cpm/mL. The prehybridization solution was removed from the blots and the hybridization solution and riboprobe were added to the blots. The hybridization was allowed to proceed overnight with gentle shaking. The total RNA blots hybridized at 63 0 C and the polyA+ RNA blots hybridized at 63 0

C.

The probed total RNA blots were washed once for 30 minutes in 2xSSC containing 0.05% SDS at 42 0 C and once for 30 minutes in 2xSSC containing 0.05% SDS at 55 0 C; twice for 30 minutes in 0.lxSSC containing 0.1% SDS at 63 0 C; three times for 30 minutes in 0.lxSSC containing 0.1%SDS and then exposed to X-ray film. The poly A+ blots were washed once for 30 minutes in a 2xSSC solution -WO 99/21997 PCT/US98/22879 containing 0.05% SDS at 63 0 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 RNA was detected in MP-1, HFF and CB23 cells. Primary 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 Semaphorin/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 supernatants from the transfected cells.

DNA encoding AHV-Sema is described 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 WC1l (Plowright, W. et al.

Nature 188:1167-1169, 1960) using PCR techniques and synthesized oligonucleotide primers whose sequences were based on the published AHV-Sema sequence. The upstream oligonucleotide primer introduced a Spe 1 site. A downstream oligonucleotide primer introduced a Not 1 site downstream of the termination codon.

The general method used to isolate the soluble AHVSema is described in Spriggs et al., J. Virology, 70:5557 (1996).

A restriction fragment containing a mutein human Fc region of immunoglobulin as described by Goodwin et al. Cell 73, 447-456, 1993 was ligated into an expression vector (pDC409) containing a murine IL-7 signal peptide and a

FLAG

T M octapeptide as described 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 murine IL-7 signal peptide and FLAGTM peptide, in 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 4m filter. Then approximately 1 L of culture supernatant was passed through a BioCad Protein A HPLC protein purification system using a 4.6 x 100 mm Protein A :WO 99/21997 PCT/US98/22879 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 purified fusions protein was neutralized as it eluted using 1M HEPES solution at pH 7.4.

Example 9 Expressing Recombinant Semaphorin Receptor Using the semaphorin receptor (VESPR) amino acid sequence of the protein purified as described in Example 5, and information derived from EST database searches and cDNAs obtained using hybridization methodologies with radiolabeled probes, also as described in Example 5, cDNA is generated and cells are transfected with the cDNA to allow expression of recombinant VESPR polypeptide.

The cDNA in DC409 expression vector, derived 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 37 0 C. The medium is replaced with 1.5 mL of medium containing 66.7 pM chloroquine and a DNA mixture containing 5 pg of cDNA encoding VESPR.

Medium containing 175iL and 25 pL of DEAE dextran is added to the cells. The cells and cDNA are incubated at 37 0 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.

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, the transfected cells are harvested, fixed in 1% paraformaldehyde, washed and used in their intact form.

Example VESPR Binding Studies In order to examine the binding characteristics of a receptor polypeptide of the present invention, binding studies were performed by subjecting cells expressing :WO 99/21997 PCT/US98/22879 membrane bound VESPR extracellular domain to the slide binding assay described in Goodwin et al. Cell 73:447-456, (1993) and Spriggs et al., J Virol 70:5557 (1996).

The pDC409 expression vector, derived 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 pDC409 expression vector through the Sal 1 and Not 1 sites, to form a DNA construct.

CV-1/EBNA cells were transfected via DEAE/Dextran with 2jig of a VESPR cDNA (encoding amino acids 19-1100) in pDC409 (Giri et al., EMBO J 13:2822, 1994). The transfected cells were cultured for 3 days and the CV-1/EBNA cell monolayers were incubated with ljtg/mL of A39R/Fc, AHVSema/Fc, or control Fc protein. Then the incubated cells were washed and incubated with 2 5 I-labeled mouse anti-human IgG (Jackson Immunoresearch, West Grove, PA). After extensive washing, the cells were fixed, dipped in photographic emulsion as described by Gearing et al., EMBO J 8:3667-3676 (1989) and developed. Positive binding was determined by the presence of exposed or darkened silver grains overlaying cells expressing VESPR that had bound Fc protein.

EXAMPLE 11 Flow Cvtometry and Inhibition Binding Studies The following describes flow cytometric analyses of CB23 cells for binding to A39R/Fc fusion protein (Example 1) and the AHVsema/Fc fusion protein (Example9). Also described 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 described in Example 2.

The flow cytometric analysis was performed by first incubating about xl106 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 washed and then incubated with phycoerythrin-conjugated Fc specific anti-human IgG in FACS buffer. The cells were washed and analyzed on a FACScan from Becton Dickinson, Bedford, MA. The results showed positive binding of AHV semaphorin and the A39R semaphorin.

Binding inhibition studies were performed by incubating about 1x10 6 CB23 cells for 30 minutes on ice in FACS buffer. The A39R/polyHis and control HIS 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 phycoerythrinconjugated Fc specific anti-human IgG in FACS buffers. The cells were washed 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 semaphorin, human tonsillar B cells were purified as described in Spriggs et al., J Exp Med 176:1543, (1992). An A39R/polyHis fusion protein was prepared as described in Example 2. A solution of A39R/polyHis fusion protein was prepared to a final A39R concentration of litg/mL and the A39R/polyHis fusion protein solution was incubated in in vitro cultures of about 105 of the purified 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 described in Example 6, was added to the fusion protein preparation prior to adding the fusion protein to the cultures, the cell aggregation was blocked. Additionally, when the A39R semaphorin was heat inactivated prior 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 cytokines, produce antibodies, or become antigen presenting cells.

Example 13 Mouse Dendritic Cells and Macrophage Aggregation with A39R Semaphorin In order to examine dendritic cell and macrophage response to A39R, mouse cell cultures were brought into contact with A39R semaphorin and the effects of the combination noted. Mouse dendritic cell cultures containing macrophages were obtained by immunizing mice with Flt3-L and cells were isolated and purified as described in Maraskovsky et al., J Exp Med 184:1953, (1996).

WO 99/21997 PCT/US98/22879 Briefly, female C57B1/6 mice were injected once daily with a solution of pg of Flt3L and 1 pig mouse serum albumin in 100 pL 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 NHICl to deplete red blood cells. The remaining cells were incubated with mAb to Thy-1, 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 enriched cells were cultured or sorted for the various cell populations.

Cells selected for sorting were stained with anti-CD1lc and anti-CD1lb and sorted for the C and D/E populations as described in Maraskovsky et al., J Exp Med 184:1953-1962, 1996.

An A39R/polyHis fusion protein was prepared as described in Example 2. An A39R/polyHis fusion protein solution was incubated in in vitro cultures at a final concentration of lig/mL with about 10 5 of the sorted or depleted mouse cells. Within 4-6 hours the cells began to aggregate. When a 10 fold molar excess of the monoclonal antibody against A39R, prepared as described in Example 6, was added to the A39R/polyHis fusion protein preparation prior to adding the fusion protein to the mouse cell cultures, the aggregation was blocked.

This work confirms that VESPR is expressed on dendritic cells and macrophages, and that the interaction between A39R and VESPR results in dendritic cell and macrophage aggregation.

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

On day 5, 1lpg/mL of A39R/polyHis fusion protein was added to the culture.

On day 6, the cells were stained with diagnostic antibodies. The results of the diagnostic antibody staining experiments showed that CD1lc', CDllb' cells (dendritic cells) expressed an increased amount of the CD69 activation antigen, thus demonstrating that the interaction of A39R semaphorin and its receptor upregulate CD69 expression.

-WO 99/21997 PCTIS98/2879 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 semaphorin and its membrane bound receptor on the regulation of the CD69 activation antigen, a transient and early expressed marker for cell activation.

Example 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 spleen cells, mice were immunized with flt3-L and dendritic cells were generated, harvested and purified as described in Example 13.

Approximately 5x10 5 cells/0.5 mL of purified, unsorted dendritic cells were incubated in modified DMEM media (500 pL at 1 x 10'/mL) in the presence of one more of the following: 20ng/mL muGM-CSF (Immunex, Seattle, WA), 20ng/mL y IFN (Genzyme, Boston, MA), 10g/mL SAC (CalBiochem, La Jolla, CA). Each cell preparation was treated additionally with 1 g/mL of A39R/polyHis fusion protein alone or in combination with 1 (ig/mL or 0.1 pg/mL of muCD40L trimer (Immunex, Seattle, WA). Cultures were incubated in humidified 37C, 10% CO 2 -in-air for 16-18 h. After incubation, the viability of each group of cultured cells was determined and supernatants 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 in particular that A39R interacts with its receptor to synergize with interferon and SAC in the production of IL-12 from unsorted mouse dendritic cells. This in vivo IL-12 induction promotes natural killer cell activation and gamma interferon production and contributes to upregulating gamma interferon sensitive cytokines.

Example 16 Testing Effects of A39R on Regulation of MHC Class II and CD86 on Monocytes The following experiment describes upregulation of MHC Class II and CD86 by the interaction of A39R with its membrane bound receptor. Peripheral blood from healthy donors was diluted 1:1 in low endotoxin PBS at pH 7.4 and room temperature. Then 35 mLs of the diluted blood was layered over 15 mLs of Isolymph :WO 99/21997 PCT/US98/22879 (Gallard and Schlesinger Industries, Inc; Carle Place, NY) and centrifuged 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, Walkersville, MD) and added to T175 flasks. The flasks had been previously coated 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 0

C,

CO, and then rinsed three times gently with 10 mL washes of low endotoxin PBS.

Adhered monocytes were harvested by incubating the cells in Enzyme Free Dissociation Buffer (Gibco, BRL) and washing the cells multiple times in PBS.

Monocytes were centrifuged at 2500 rpm for 5 minutes, counted, and set up in 24 well dishes at 5 x 105 cells/well in 1 mL. The cultures were 95% pure.

Purified 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 dendritic cell-like phenotype. On day 7-9, cultures were treated with 1 ig/mL A39R/polyHis or a control polyHis containing protein, and the next day cells and supernatants were harvested for analyses.

In flow cytometric experiments for examining monocyte-derived dendritic cell surface markers, cells were stained with conjugated mAbs directed against specific proteins. The staining showed that for a majority of the peripheral blood donors 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 dendritic cells, their downregulation suggests an immunosuppressive effect of the interaction of A39R with its receptor on this cell population.

Example 17 Upregulation of CD54 The following describes the effect of the interaction between A39R semaphorin and its receptor on purified monocytes and more particularly, the impact of CD54 expression on monocytes after incubation with a semaphorin. Freshly isolated monocytes were purified from peripheral blood donors as described in 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 all donors tested, the level of CD54 surface expression was enhanced in the presence -WO 99/21997 PCT/US98/22879 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 human monocytes.

Example 18 Cytokine Induction from Freshly Isolated Human Monocytes Freshly isolated human monocytes were purified as described in Example 16, and cultured as described in Example 17. After the overnight incubation with A39R/polyHis, monocyte supernatants were examined for the presence of proinflammatory cytokines. In all donors tested, IL-6 and IL-8 was induced by A39R protein. 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 against A39R.

The results of this experiment 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 Monocvte Aggregation Studies In order to examine human monocyte response to the interaction of a semaphorin to its receptor on monocytes, monocytes were purified as described in Example 17 and an A39R/polyHIS fusion protein was prepared as' described in Example 2. The fusion protein and purified, 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 result of CD54 upregulation. However, other factors may contribute to the aggregation as well.

This work confirms that the semaphorin receptor of the present invention is expressed on monocytes and that the interaction between A39R and VESPR results in monocyte aggregation. Similar to B cells, monocytes aggregation is indicative of their activation.

.'x.ample Monoclonal Antiodies to VI:;SIT' This cxa.unpi illustrateis ;i method for prcp;ring antib-odics to VESPR polypcplidc.s. IPurifed VEISPR polypcptide is prepared as described in Example 9.

The puificd protein is used to generate antibodies against VEISPR as described in U.S. Patent 4,411,993. Briefly, mice are immunized at 0, 2 and 6 weeks with 10 pg with V\ESPR. The primary immunization is prepared with TITERMAX adjuvant, from Vaxcell, Inc., and subsequent immunizations are prepared with incomplete Freund's adjuvant (EFA). At 11 weeks, the mice are IV boosted with 3-4 tg VESPR in PBS. Three days after the IV boost, splenocytes are harvested and fused with an Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution. Hybridoma supernatants are screened for VESPR antibodies by dot blot assay against VESPR and an irrelevant Fc protein.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in i this specification solely for the purpose of providing a context for the present invention.

It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

EDITORIAL NOTE NO. 12047/99 SEQUENCE LISTING PAGE 1 TO 22 FOLLOW PAGE 42.

:WO 99/21997 PCT/US98/22879 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Immunex Corporation, Melanie K. Spriggs, Michael R. Comeau, Robert F. DuBose, Richard S. Johnson (ii) TITLE OF INVENTION: VIRAL ENCODED SEMAPHORIN PROTEIN RECEPTOR DNA AND POLYPEPTIDES (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Janis C. Henry STREET: 51 University St.

CITY: Seattle STATE: WA COUNTRY: US ZIP: 98101 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: to be assigned-- FILING DATE: 28-OCT-98

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/958,598 (converted to a Provisional, see below) FILING DATE: 28-OCT-1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: to be assigned-- (USSN 08/958,598 conversion to Provisional application) FILING DATE: 28-OCT-1997

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Henry, Janis C REGISTRATION NUMBER: 34,347 REFERENCE/DOCKET NUMBER: 2631-WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (206)470-4189 TELEFAX: (206)233-0644 INFORMATION FOR SEQ ID NO:1: WO 99/21997 PCT[US98/22879 SEQUENCE CHARACTERISTICS: LENGTH: 4707 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..4707 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATG

Met 1 GAG GTC TCC Glu Val Ser

CGG

Arg 5 AGG AAG GCG CCG Arg Lys Ala Pro

CCG

Pro 10 CGC CCC CCG CGC Arg Pro Pro Arg CCC GCA Pro Ala GCG CCA CTG Ala Pro Leu CGG GGC GCG Arg Gly Ala CTG CTC GCC TAT Leu Leu Ala Tyr CTG GCA CTG GCG Leu Ala Leu Ala GCT CCC GGC Ala Pro Gly ATC GGA GCC Ile Gly Ala GAC GAG CCC GTG Asp Glu Pro Val CGG TCG GAG CAA Arg Ser Glu Gin ATC GCG Ile Ala GCG AGC CAG GAG Ala Ser Gln Glu

GAC

Asp GGC GTG TTT GTG Gly Val Phe Val AGC GGC AGC TGC Ser Gly Ser Cys

CTG

Leu GAC CAG CTG GAC Asp Gln Leu Asp AGC CTG GAG CAC Ser Leu Glu His

AGC

Ser CTC TCG CGC CTG Leu Ser Arg Leu 240 CGG GAC CAA GCG Arg Asp Gln Ala

GGC

Gly AAC TGC ACA GAG Asn Cys Thr Glu

CCG

Pro 90 GTC TCG CTG GCG Val Ser Leu Ala CCC CCC Pro Pro 288 GCG CGG CCC Ala Arg Pro CGC GAG GGG Arg Glu Gly 115 CCC GGG AGC AGC Pro Gly Ser Ser AGC AAG CTG CTG Ser Lys Leu Leu CTG CCC TAC Leu Pro Tyr 110 GGC TGG ACC Gly Trp Thr GCG GCC GGC CTC Ala Ala Gly Leu

GGG

Gly 120 GGG CTG CTG CTC Gly Leu Leu Leu

ACC

Thr 125 TTC GAC Phe Asp 130 CGG GGC GCC TGC Arg Gly Ala Cys

GAG

Glu 135 GTG CGG CCC CTG Val Arg Pro Leu

GGC

Gly 140 AAC CTG AGC CGC Asn Leu Ser Arg AAC TCC CTG CGC AAC GGC ACC GAG GTG GTG TCG TGC CAC CCG CAG GGC Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro Gln Gly :WO 99/21997 PCT/US98/22879 155 160 TCG AGG GCC GGC Ser Thr Ala Gly

GTG

Val 165 GTG TAC CGC GCG Val Tyr Arg Ala GGG AAG AAC GGC Arg Asn Asn Arg TGG TAG Trp Tyr 175 528 CTG GCG GTG Leu Ala Val CGG TGC AAC Arg Cys Asn 195

GCC

Ala 180 GCC AGC TAG GTG Ala Thr Tyr Val

CTG

Leu 185 GCT GAG CCG GAG Pro Glu Pro Glu ACG GCG AGC Thr Ala Ser 190 GCG CTC AAG Ala Leu Lys 576 CCC GGG GCA TCC Pro Ala Ala Ser

GAG

Asp 200 GAC GAG AGG GCG His Asp Thr Ala GAG ACG Asp Thr 210 GAG GGG CGG AGC Olu Gly Arg Ser

CTG

Leu 215 GCC ACG CAG GAG Ala Thr Gin Glu

CTG

Leu 220 CGG CGC CTC AAG Oly Arg Leu Lys

GTG

Leu 225 TGG GAG GGG GCG Cys Glu Gly Ala

GGC

Cly 230 AGC CTG CAC TTC Ser Leu His Phe

GTG

Val 235 GAC GCC TTT CTC Asp Ala Phe Leu

TGG

Trp 240 AAC GGC AGG ATC Asn Cly Ser Ile

TAC

Tyr 245 TTC CCC TAG TAG Phe Pro Tyr Tyr

CCC

Pro 250 TAG AAC TAT ACG Tyr Asn Tyr Thr AGC GGC Ser Gly 255 GCT GCC ACC Ala Ala Thr CTG CTG TTC Val Leu Phe 275

GGC

Gly 260 TGC CCC AGC ATG Trp Pro Ser Met CGC ATC GGG CAG Arg Ile Ala Gin AGC AGC GAG Ser Thr Glu 270 GAG GGC CAG GCA Gin Cly Gin Ala

TCC

Ser 280 CTC GAG TGC GGC CAC GGC CAC CCC Leu Asp Gys Oly His Oly His Pro 285 GAG GGG Asp Gly 290 CGC CGC CTG CTC Arg Arg Leu Leu TCC TCG AGC GTA Ser Ser Ser Leu

GTG

Val 300 GAG GCC CTG GAG Olu Ala Leu Asp

GTG

Va1 305 TGG GCG GGA GTG Trp Ala Gly Val

TTG

Phe 310 AGC GCG GCC GCT Ser Ala Ala Ala

GGA

Cly 315 GAG GGC CAG GAO Glu Oly Gin Glu

CGG

Arg 320 CGC TCC CCC ACC Arg Ser Pro Thr

ACC

Thr 325 ACG GCG CTC TGC Thr Ala Leu Gys

CTC

Leu 330 TTG AGA ATO ACT GAG ATG Phe Arg Met Ser Glu Ile 335 1008 GAG GCG CGC Gin Ala Arg CAC TGC AAA His Gys Lys 355 AAC AGG GTC AGG Lys Arg Val Ser GAG TTG AAC ACG Asp Phe Lys Thr GCC GAG AGC Ala Glu Ser 350 ATC GCA TCA Ile Ala Ser 1056 1104 GAA GGG OAT GAA Glu Cly Asp Gin

CCT

Pro 360 OAA AGA GTC CAA Glu Arg Val Gin

CCA

Pro 365 TCT ACC Ser Thr 370 TTG ATC CAT TCC Leu Ile His Ser CTG AGA TCC GTT TAT GGC ACC GTC GTA Leu Thr Ser Val Tyr Oly Thr Val Val 380 1152 WO 99/21997 PCT/US98/22879

ATG

Met 385 AAC AGG ACT GTT Asn Arg Thr Val

TTA

Leu 390 TTC TTG GGG Phe Leu Gly ACT GGA GAT GGC CAG TTA Thr Gly 395 Asp Gly Gin Leu

CTT

Leu 400 1200 1248 AAG GTT ATT CTT Lys Val Ile Leu GAG AAT TTG ACT Glu Asn Leu Thr

TCA

Ser 410 AAT TGT CCA GAG GTT ATC Asn Cys Pro Glu Val Ile 415 TAT GAA ATT Tyr Glu Ile CCT GTG AAG Pro Val Lys 435

AAA

Lys 420 GAA GAG ACA CCT Glu Glu Thr Pro

GTT

Val 425 TTC TAC AAA CTC Phe Tyr Lys Leu GTT CCT GAT Val Pro Asp 430 GAG GTG AGG Glu Val Arg 1296 1344 AAT ATC TAC ATT Asn Ile Tyr Ile

TAT

Tyr 440 CTA ACA GCT GGG Leu Thr Ala Gly

AAA

Lys 445 AGA ATT Arg Ile 450 CGT GTT GCA AAC Arg Val Ala Asn AAT AAA CAT AAA Asn Lys His Lys TGT TCG GAG TGT Cys Ser Glu Cys 1392 1440

TTA

Leu 465 ACA GCC ACA GAC Thr Ala Thr Asp

CCT

Pro 470 CAC TGC GGT TGG His Cys Gly Trp

TGC

Cys 475 CAT TCG CTA CAA His Ser Leu Gin TGC ACT TTT CAA Cys Thr Phe Gin

GGA

Gly 485 GAT TGT GTA CAT Asp Cys Val His

TCA

Ser 490 GAG AAC TTA GAA Glu Asn Leu Glu AAC TGG Asn Trp 495 1488 CTG GAT ATT Leu Asp Ile CGA AGC AGT Arg Ser Ser 515

TCG

Ser 500 TCT GGA GCA AAA Ser Gly Ala Lys

AAG

Lys 505 TGC CCT AAA ATT Cys Pro Lys Ile CAG ATA ATT Gin Ile Ile 510 AGC TTC TCT Ser Phe Ser 1536 1584 AAA GAA AAG ACT Lys Glu Lys Thr

ACA

Thr 520 GTG ACT ATG GTG Val Thr Met Val

GGA

Gly 525 CCA AGA Pro Arg 530 CAC TCA AAG TGC His Ser Lys Cys

ATG

Met 535 GTG AAG AAT GTG Val Lys Asn Val TCT AGC AGG GAG Ser Ser Arg Glu 1632 1680

CTC

Leu 545 TGC CAG AAT AAA Cys Gin Asn Lys

AGT

Ser 550 CAG CCC AAC CGG Gin Pro Asn Arg

ACC

Thr 555 TGC ACC TGT AGC Cys Thr Cys Ser CCA ACC AGA GCA Pro Thr Arg Ala TAC AAA GAT GTT Tyr Lys Asp Val

TCA

Ser 570 GTT GTC AAC GTG Val Val Asn Val ATG TTC Met Phe 575 1728 TCC TTC GGT Ser Phe Gly TCA TCA TTA Ser Ser Leu 595

TCT

Ser 580 TGG AAT TTA TCA Trp Asn Leu Ser

GAC

Asp 585 AGA TTC AAC TTT Arg Phe Asn Phe ACC AAC TGC Thr Asn Cys 590 TGC GCG TGG Cys Ala Trp 1776 AAA GAA TGC CCA GCA TGC GTA GAA ACT Lys Glu Cys Pro Ala Cys Val Glu Thr 600

GGC

Gly 605 1824 TGT AAA AGT GCA AGA AGG TGT ATC CAC CCC TTC ACA GCT TGC GAC CCT Cys Lys Ser Ala Arg Arg Cys Ile His Pro Phe Thr Ala Cys Asp Pro 1872 :WO 99/21997 PCT/US98/22879

TCT

Ser 625 GAT TAT GAG AGA Asp Tyr Glu Arg CAG GAA CAG TGT Gin Glu Gin Cys

CCA

Pro 635 GTG GCT GTC GAG Val Ala Val Glu

AAG

Lys 640 1920 1968 ACA TCA GGA GGA Thr Ser Gly Gly

GGA

Gly 645 AGA CCC AAG GAG Arg Pro Lys Glu AAG GGG AAC AGA Lys Gly Asn Arg ACC AAC Thr Asn 655 CAG GCT TTA Gin Ala Leu TCG ACA TTA Ser Thr Leu 675

CAG

Gin 660 GTC TTC TAC ATT Val Phe Tyr Ile

AAG

Lys 665 TCC ATT GAG CCA Ser Ile Glu Pro CAG AAA GTA Gin Lys Val 670 AAC TTT ACC Asn Phe Thr 2016 2064 GGG AAA AGC AAC Gly Lys Ser Asn

GTG

Val 680 ATA GTA ACG GGA Ile Val Thr Gly CGG GCA Arg Ala 690 TCG AAC ATC ACA Ser Asn Ile Thr ATC CTG AAA GGA Ile Leu Lys Gly AGT ACC TGT GAT Ser Thr Cys Asp 2112

AAG

Lys 705 GAT GTG ATA CAG Asp Val Ile Gin

GTT

Val 710 AGC CAT GTG CTA AAT GAC ACC CAC ATG Ser His Val Leu Asn Asp Thr His Met 715 2160 TTC TCT CTT CCA Phe Ser Leu Pro

TCA

Ser 725 AGC CGG AAA GAA Ser Arg Lys Glu AAG GAT GTG TGT Lys Asp Val Cys ATC CAG Ile Gin 735 2208 TTT GAT GGT Phe Asp Gly CTG CCA CAT Leu Pro His 755

GGG

Gly 740 AAC TGC TCT TCT Asn Cys Ser Ser

GTG

Val 745 GGA TCC TTA TCC Gly Ser Leu Ser TAC ATT GCT Tyr Ile Ala 750 ATC AGT GGT Ile Ser Gly 2256 2304 TGT TCC CTT ATA Cys Ser Leu Ile

TTT

Phe 760 CCT GCT ACC ACC Pro Ala Thr Thr GGT CAA Gly Gin 770 AAT ATA ACC ATG Asn Ile Thr Met GGC AGA AAT TTT Gly Arg Asn Phe GTA ATT GAC AAC Val Ile Asp Asn

TTA

Leu 785 ATC ATT TCA CAT Ile Ile Ser His

GAA

Glu 790 TTA AAA GGA AAC Leu Lys Gly Asn

ATA

Ile 795 AAT GTC TCT GAA Asn Val Ser Glu

TAT

Tyr 800 2352 2400 2448 TGT GTG GCG ACT Cys Val Ala Thr

TAC

Tyr 805 TGC GGG TTT TTA Cys Gly Phe Leu

GCC

Ala 810 CCC AGT TTA AAG Pro Ser Leu Lys AGT TCA Ser Ser 815 AAA GTG CGC Lys Val Arg TTG GAT TGT Leu Asp Cys 835

ACG

Thr 820 AAT GTC ACT GTG Asn Val Thr Val

AAG

Lys 825 CTG AGA GTA CAA Leu Arg Val Gin GAC ACC TAC Asp Thr Tyr 830 2496 GGA ACC CTG CAG Gly Thr Leu Gin TAT CGG Tyr Arg 840 GAG GAC CCC AGA TTC ACG GGG Glu Asp Pro Arg Phe Thr Gly 845 2544 .WO 99/21997 PCT/US98/22879 TAT CGG Tyr Arg 850 GTG GAA TCC GAG Val Glu Ser Glu

GTG

Val 855 GAC ACA GAA CTG Asp Thr Glu Leu

GAA

Glu 860 GTG AAA ATT CAA Val Lys Ile Gin

AAA

Lys 865 GAA AAT GAC AAC Glu Asn Asp Asn AAT ATT TCC AAA Asn Ile Ser Lys

AAA

Lys 875 GAC ATT GAA ATT Asp Ile Glu Ile

ACT

Thr 880 2592 2640 2688 CTC TTC CAT GGG Leu Phe His Gly AAT GGG CAA TTA Asn Gly Gin Leu

AAT

Asn 890 TGC AGT TTT GAA Cys Ser Phe Glu AAT ATT Asn Ile 895 ACT AGA AAT Thr Arg Asn AAG ACT GCA Lys Thr Ala 915

CAA

Gin 900 GAT CTT ACC ACC Asp Leu Thr Thr

ATC

Ile 905 CTT TGC AAA ATT Leu Cys Lys Ile AAA GGC ATC Lys Gly Ile 910 CGG GTC AAG Arg Val Lys 2736 2784 AGC ACC ATT GCC Ser Thr Ile Ala TCT TCT AAG AAA Ser Ser Lys Lys

GTT

Val 925 CTG GGA Leu Gly 930 AAC CTG GAG CTC Asn Leu Glu Leu GTC GAG CAG GAG Val Glu Gin Glu

TCA

Ser 940 GTT CCT TCC ACA Val Pro Ser Thr 2832 2880 TGG Trp 945 TAT TTT CTG ATT Tyr Phe Leu Ile

GTG

Val 950 CTC CCT GTC TTG Leu Pro Val Leu

CTA

Leu 955 GTG ATT GTC ATT Val Ile Val Ile GCG GCC GTG GGG Ala Ala Val Gly ACC AGG CAC AAA Thr Arg His Lys

TCG

Ser 970 AAG GAG CTG AGT Lys Glu Leu Ser CGC AAA Arg Lys 975 2928 CAG AGT CAA Gin Ser Gin CGT GAC GGC Arg Asp Gly 995 AGT TTT GGA Ser Phe Gly 1010

CAA

Gin 980 CTA GAA TTG CTG Leu Glu Leu Leu

GAA

Glu 985 AGC GAG CTC Ser Glu Leu CGG AAA GAG ATA Arg Lys Glu Ile 990 GAT GTG GTT GAT Asp Val Val Asp 1005 TTT GCT GAG CTG Phe Ala Glu Leu CAG ATG GAT AAA TTG Gin Met Asp Lys Leu 1000 2976 3024 3072 ACT GTT CCC Thr Val Pro TTC CTT GAC TAC AAA Phe Leu Asp Tyr Lys 1015 CAT TTT His Phe 1020 GCT CTG AGA Ala Leu Arg ACT TTC TTC CCT GAG Thr Phe Phe Pro Glu 1025 TCA GGT GGC TTC ACC Ser Gly Gly Phe Thr 1030 CAC ATC His Ile 1035 TTC ACT GAA Phe Thr Glu

GAT

Asp 1040 3120 ATG CAT AAC AGA GAC GCC AAC GAC AAG Met His Asn Arg Asp Ala Asn Asp Lys 1045 AAT GAA AGT CTC ACA Asn Glu Ser Leu Thr 1050 GCT TTG Ala Leu 1055 3168 GAT GCC CTA ATC TGT AAT AAA AGC Asp Ala Leu Ile Cys Asn Lys Ser 1060 TTT CTT GTT ACT GTC Phe Leu Val Thr Val 1065 ATC CAC ACC Ile His Thr 1070 3216 CTT GAA AAG CAG AAG AAC TTT TCT GTG AAG GAC AGG TGT CTG TTT GCC Leu Glu Lys Gin Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Ala 3264 WO 99/21997 PCT/S98/22879 1075 1080 1085 TCC TTC CTA Ser Phe Leu 1090 ACC ATT GCA CTG CAA Thr Ile Ala Leu Gin 1095 ACC AAG CTG GTC TAC Thr Lys Leu Val Tyr 1100 CTG ACC AGC Leu Thr Ser 3312 ATC CTA Ile Leu 1105 GAG GTG CTG ACC AGG Glu Val Leu Thr Arg 1110 GAC TTG ATG GAA CAG Asp Leu Met Glu Gin 1115 TGT AGT AAC ATG Cys Ser Asn Met 1120 3360 CAG CCG AAA Gin Pro Lys CTC ACA AAC Leu Thr Asn CTC ATG CTG Leu Met Leu 1125 AGA CGC ACG GAG TCC Arg Arg Thr Glu Ser 1130 GTC GTC GAA AAA CTC Val Val Glu Lys Leu 1135 3408 3456 TGG ATG Trp Met 1140 TCC GTC TGC Ser Val Cys CTT TCT Leu Ser 1145 GGA TTT CTC Gly Phe Leu GTC GGA GAG CCC TTC TAT TTG CTG GTG ACG ACT CTG AAC Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn CGG GAG ACT Arg Glu Thr 1150 CAG AAA ATT Gin Lys Ile TAC ACA CTT Tyr Thr Leu 3504 1155 1160 AAC AAG GGT CCC GTG GAT Asn Lys Gly Pro Val Asp 1170

GTA

Val 1175 ATC ACT TGC AAA GCC Ile Thr Cys Lys Ala 116!

CTG

Leu 0 3552 118( AAT GAA GAC TGG CTG Asn Glu Asp Trp Leu 1185 TTG TGG Leu Trp 1190 CAG GTT CCG GAA TTC AGT ACT GTG Gin Val Pro Glu Phe Ser Thr Val 1195

GCA

Ala 1200 3600 TTA AAC GTC GTC Leu Asn Val Val TTT GAA AAA ATC CCG Phe Glu Lys Ile Pro 1205 GAA AAC GAG AGT GCA Glu Asn Glu Ser Ala 1210 GAT GTC Asp Val 1215 3648 TGT CGG AAT Cys Arg Asn ATT TCA GTC AAT GTT Ile Ser Val Asn Val 1220 CTC GAC Leu Asp 1225 TGT GAC ACC Cys Asp Thr ATT GGC CAA Ile Gly Gin 1230 3696 3744 GCC AAA GAA AAG Ala Lys Glu Lys 1235 ATT TTC CAA Ile Phe Gin GCA TTC Ala Phe 1240 TTA AGC AAA Leu Ser Lys AAT GGC TCT CCT Asn Gly Ser Pro 1245 TAT GGA CTT CAG CTT AAT GAA ATT GGT CTT GAG Tyr Gly Leu Gin Leu Asn Glu Ile Gly Leu Glu 1250 1255 CTT CAA ATG GGC ACA Leu Gin Met Gly Thr 1260 3792 CGA CAG AAA GAA CTT Arg Gin Lys Glu Leu 1265 CTG GAC ATC GAC AGT Leu Asp Ile Asp Ser 1270 TCC TCC GTG ATT CTT Ser Ser Val Ile Leu 1275

GAA

Glu 1280 3840 GAT GGA ATC ACC AAG CTA AAC ACC ATT GGC CAC TAT GAG ATA TCA AAT Asp Gly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn 1285 1290 1295 GGA TCC ACT ATA AAA GTC TTT AAG AAG ATA GCA AAT TTT ACT TCA GAT Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp 1300 1305 1310 3888 3936 WO 99/21997 PCT/US98/22879 GTG GAG TAC TCG Val Glu Tyr Ser 1315 GAT GAC CAC TGC CAT Asp Asp His Cys His 1320 TTG ATT TTA CCA GAT TCG GAA Leu Ile Leu Pro Asp Ser Glu 1325 3984 GCA TTC CAA Ala Phe Gin 1330 GAT GTG CAA GGA AAG Asp Val Gin Gly Lys 1335 AGA CAT CGA GGG AAG Arg His Arg Gly Lys 1340 CAC AAG TTC His Lys Phe 4032 AAA GTA Lys Val 1345 AAA GAA ATG TAT CTG Lys Glu Met Tyr Leu 1350 ACA AAG CTG CTG TCG Thr Lys Leu Leu Ser 1355 ACC AAG GTG GCA Thr Lys Val Ala 1360 4080 4128 ATT CAT TCT GTG Ile His Ser Val CTT GAA Leu Glu 1365 AAA CTT TTT Lys Leu Phe AGA AGC Arg Ser 1370 ATT TGG AGT Ile Trp Ser TTA CCC Leu Pro 1375 AAC AGC AGA Asn Ser Arg GCT CCA Ala Pro 1380 TTT GCT ATA Phe Ala Ile AAA TAC TTT TTT GAC Lys Tyr Phe Phe Asp 1385 TTT TTG GAC Phe Leu Asp 1390 GTA CAT ATT Val His Ile 4176 4224 GCC CAG GCT GAA Ala Gin Ala Glu 1395 AAC AAA AAA Asn Lys Lys ATC ACA GAT CCT GAC Ile Thr Asp Pro Asp 1400

GTC

Val 1405 TGG AAA Trp Lys 1410 AAC CCT Asn Pro 1425 ACA AAC AGC CTT Thr Asn Ser Leu CCT CTT CGC TTC TGG Pro Leu Arg Phe Trp 1415 GTA AAC ATC CTG AAG Val Asn Ile Leu Lys 1420 CAG TTT GTC G1n Phe Val TTT GAC ATT AAG AAG Phe Asp Ile Lys Lys 1430 ACA CCA Thr Pro 1435 CAT ATA GAC His Ile Asp

GGC

Gly 1440 4272 4320 4368 4416 TGT TTG TCA GTG Cys Leu Ser Val ATT GCC CAG GCA TTC Ile Ala Gin Ala Phe 1445 ATG GAT GCA TTT TCT Met Asp Ala Phe Ser 1450 CTC ACA Leu Thr 1455 GAG CAG CAA Glu Gin Gin CTA GGG AAG GAA GCA CCA ACT AAT AAG CTT Leu Gly Lys Glu Ala Pro Thr Asn Lys Leu 1460 1465 CTC TAT GCC Leu Tyr Ala 1470 AAG GAT ATC Lys Asp Ile 1475 CCA ACC TAC AAA Pro Thr Tyr Lys GAA GAA GTA AAA TCT Glu Glu Val Lys Ser 1480 TAT TAC AAA GCA Tyr Tyr Lys Ala 1485 4464 ATC AGG Ile Arg 1490 GAT TTG CCT CCA TTG TCA TCC TCA GAA Asp Leu Pro Pro Leu Ser Ser Ser Glu ATG GAA GAA TTT TTA Met Glu Glu Phe Leu 1500 1495 ACT CAG GAA TCT AAG Thr Gin Glu Ser Lys 1505 AAA CAT GAA AAT GAA TTT AAT GAA GAA GTG Lys His Glu Asn Glu Phe Asn Glu Glu Val 1510 1515

GCC

Ala 1520 4512 4560 4608 TTG ACA GAA ATT Leu Thr Glu Ile

TAC

Tyr 1525 AAA TAC ATC GTA AAA TAT TTT GAT GAG ATT CTA Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile Leu 1530 1535 AAT AAA CTA GAA AGA GAA CGA GGG CTG GAA GAA GCT CAG AAA CAA CTC Asn Lys Leu Glu Arg Glu Arg Gly Leu Glu Glu Ala Gin Lys Gin Leu 4656 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 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1569 amino acids TYPE: amino acid TOPOLOGY: linear (ii) 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 1 5 10 Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly 25 Arg Gly Ala Asp Glu Pro Val Trp Arg Ser Glu Gln Ala Ile Gly Ala 40 Ile Ala Ala Ser Gin Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys 55 Leu Asp Gln Leu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr 70 75 Arg Asp Gin Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro 90 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 Gln 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 Ile Ala Leu Lys 9 :WO 99/21997 :W99/2997PCTIUS98/22879 Asp Leu 225 Asn Ala Val Asp Val 305 Arg Gin His Ser Met 385 LAys Tyr Pro Arg Leu 465 Cys Thr 210 Cys Gly Ala Leu Gly 290 Trp Ser Ala Cys Thr 370 Asn Val1 Glu Val1 Ile 450 Thr Thr 195 Giu Glu Ser Thr Phe 275 Arg Ala Pro Arg Lys 355 Leu Arg Ile Ile Lys 435 Arg Ala Phe 200 205 Gly Arg Ser Leu Ala Thr Gin Glu Leu Gly Arg Leu Lys Gly Ile Gly 260 Gin Arg Gly Thr Aia 340 Giu Ile Thr Leu Lys 420 Asn Vai Thr Gin Ser 500 Ala Tyr 245 Trp Gly Leu Val Thr 325 Lys Gly His Val1 Gly 405 Giu Ile Ala Asp Gly 485 Gly 230 Phe Pro Gln Leu Phe 310 Thr Arg Asp Ser Leu 390 Giu Glu Tyr Asn Pro 470 Asp 215 Ser Pro Ser Ala Leu 295 Ser Al a Val1 Gin Asp 375 Phe Asn Thr Ile Cys 455 His Cys Leu Tyr Met Ser 280 Ser Ala Leu Ser Pro 360 Leu Leu Leu Pro Tyr 440 Asn Cys Val His Tyr Ala 265 Leu Ser Ala Cys Trp 345 Giu Thr Gly Thr Val1 425 Leu Lys Gly His Lys 505 Phe Pro 250 Arg Asp Ser Ala Leu 330 Asp Arg Ser Thr Ser 410 Phe Thr His Trp Ser 490 Cys Val 235 Tyr Ile Cys Leu Gly 315 Phe Phe Val1 Vai Gly 395 Asn Tyr Ala Lys Cys 475 Giu Pro 220 Asp Ala Asn Tyr Ala Gin Gly His 285 Val Giu 300 Giu Gly Arg Met Lys Thr Gin Pro 365 Tyr Gly 380 Asp Gly Cys Pro Lys Leu Gly Lys 445 Ser Cys 460 His Ser Asn Leu Lys Ile Phe Thr Ser 270 Gly Ala Gin Ser Al a 350 Ile Thr Gln Glu Val1 430 Giu Ser Leu Glu Gin 510 Leu Ser 255 Thr His Leu Giu Glu 335 Glu Ala Val Leu Val 415 Pro Val Giu Gin Asn 495 Ile Trp 240 Gly Giu Pro Asp Arg 320 Ile Ser Ser Val1 Leu 400 Ile Asp Arg Cys Arg 480 Trp, Ile Leu Asp Ile Ser Giy Ala Lys -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 Ile 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 Ile 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 Ile Lys Ser Ile Glu Pro Gin Lys Val 660 665 670 Ser Thr Leu Gly Lys Ser Asn Val Ile Val Thr Gly Ala Asn Phe Thr 675 680 685 Arg Ala Ser Asn Ile Thr Met Ile Leu Lys Gly Thr Ser Thr Cys Asp 690 695 700 Lys Asp Val Ile 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 Ile Gin 725 730 735 Phe Asp Gly Gly Asn Cys Ser Ser Val Gly Ser Leu Ser Tyr Ile Ala 740 745 750 Leu Pro His Cys Ser Leu Ile Phe Pro Ala Thr Thr Trp Ile Ser Gly 755 760 765 Gly Gin Asn Ile Thr Met Met Gly Arg Asn Phe Asp Val Ile Asp Asn 770 775 780 Leu Ile Ile Ser His Glu Leu Lys Gly Asn Ile 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 :WO99/21997 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 Giu Asp Pro Arg Phe Thr Gly 835 840 845 Tyr Arg Val Glu Ser Giu Val Asp Thr Giu Leu Giu Val Lys Ile Gin 850 855 860 Lys Giu Asn Asp Asn Phe Asn Ile Ser Lys Lys Asp Ile Giu Ile Thr 865 870 875 880 Leu Phe His Giy Giu Asn Gly Gin Leu Asn Cys Ser Phe Giu Asn Ile 885 890 895 Thr Arg Asn Gin Asp Leu Thr Thr Ile Leu Cys Lys Ile Lys Gly Ile 900 905 910 Lys Thr Ala Ser Thr Ile Ala Asn Ser Ser Lys Lys Val Arg Val Lys 915 920 925 Leu Gly Asn Leu Giu Leu Tyr Val Glu Gin Glu Ser Vai Pro Ser Thr 930 935 940 Trp Tyr Phe Leu Ile Val Leu Pro Val Leu Leu Vai Ile Val Ile Phe 945 950 955 960 Ala Ala Val Gly Val Thr Arg His Lys Ser Lys Giu Leu Ser Arg Lys 965 970 975 Gin Ser Gin Gin Leu Giu Leu Leu Glu Ser Giu Leu Arg Lys Glu Ile 980 985 990 Arg Asp Gly Phe Aia Giu Leu Gin Met Asp Lys Leu Asp Val Val Asp 995 1000 1005 Ser Phe Giy Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg 1010 1015 1020 Thr Phe Phe Pro Giu Ser Gly Gly Phe Thr His Ile Phe Thr Giu Asp 1025 1030 1035 1040 Met His Asn Arg Asp Ala Asn Asp Lys Asn Giu Ser Leu Thr Ala Leu 1045 1050 1055 Asp Ala Leu Ile Cys Asn Lys Ser Phe Leu Val Thr Val Ile His Thr 1060 1065 1070 Leu Giu Lys Gin Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Aia 1075 1080 1085 Ser Phe Leu Thr Ile Ala Leu Gin Thr Lys Leu Val Tyr Leu Thr Ser 1090 1095 1100 Ile Leu Giu Val Leu Thr Arg Asp Leu Met Giu Gin Cys Ser Asn Met 1105 1110 1115 1120 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 Ile 1155 1160 1165 Asn Lys Gly Pro Val Asp Val Ile 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 Ile Pro Glu Asn Glu Ser Ala Asp Val 1205 1210 1215 Cys Arg Asn Ile Ser Val Asn Val Leu Asp Cys Asp Thr Ile Gly Gin 1220 1225 1230 Ala Lys Glu Lys Ile Phe Gin Ala Phe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 Tyr Gly Leu Gin Leu Asn Glu Ile Gly Leu Glu Leu Gin Met Gly Thr 1250 1255 1260 Arg Gin Lys Glu Leu Leu Asp Ile Asp Ser Ser Ser Val Ile Leu Glu 1265 1270 1275 1280 Asp Gly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn 1285 1290 1295 Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp 1300 1305 1310 Val Glu Tyr Ser Asp Asp His Cys His Leu Ile 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 Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro 1365 1370 1375 Asn Ser Arg Ala Pro Phe Ala Ile Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 Ala Gin Ala Glu Asn Lys Lys Ile Thr Asp Pro Asp Val Val His Ile 1395 1400 1405 Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn Ile Leu Lys 1410 1415 1420 -WO 99/21997 PCT/US98/22879 Asn Pro Gin Phe Val Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly 1425 1430 1435 1440 Cys Leu Ser Val Ile 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 Ile Pro Thr Tyr Lys Glu Glu Val Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 Ile 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 Ile Tyr Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile 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 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGTCACTAGT ATCGAATGGC ATAAGTTTGA A 31 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 99/21997 PCT/US98/22879 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GACAGCGGCC GCCTATTACA TTTTAAGTAT TTT 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

NAME/KEY:

LOCATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID GCGGGACTCA GAGTCACC 18 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGATCCTAAT ACGACTCACT ATAGGGAGGA AACCACTCCG AAC 43 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1983 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -WO 99/21997 PCTIUS98/22879 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1983 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ATG

Met 1 TTC CAT GTT TCT TTT AGA TAT ATC Phe His Val Ser Phe Arg Tyr Ile 5 GGA ATT CCT CCA Gly Ile Pro Pro CTG ATC Leu Ile CTT GTT CTG Leu Val Leu AAA AGA TCT Lys Arg Ser CCT GTC ACT AGC Pro Val Thr Ser

TCT

Ser GAC TAC AAA GAT Asp Tyr Lys Asp GAC GAT GAT Asp Asp Asp TGT GAC AAA ACT Cys Asp Lys Thr CAC ACA TGC CCA CCG TGC CCA GCA CCT His Thr Cys Pro Pro Cys Pro Ala Pro 40 GTC TTC CTC TTC CCC CCA AAA CCC AAG Val Phe Leu Phe Pro Pro Lys Pro Lys GAA GCC Glu Ala GAG GGC GCG CCG Glu Gly Ala Pro

TCA

Ser 55

GAC

Asp ACC CTC ATG ATC Thr Leu Met Ile

TCC

Ser 70 CGG ACC CCT GAG Arg Thr Pro Glu

GTC

Val 75 ACA TGC GTG GTG Thr Cys Val Val

GTG

Val GAC GTG AGC CAC Asp Val Ser His

GAA

Glu GAC CCT GAG GTC Asp Pro Glu Val TTC AAC TGG TAC Phe Asn Trp Tyr GTG GAC Val Asp GGC GTG GAG Gly Val Glu AAC AGC ACG Asn Ser Thr 115

GTG

Val 100 CAT AAT GCC AAG His Asn Ala Lys

ACA

Thr 105 AAG CCG CGG GAG Lys Pro Arg Glu GAG CAG TAC Glu Gin Tyr 110 CAC CAG GAC His Gin Asp 336 384 TAC CGT GTG GTC Tyr Arg Val Val GTC CTC ACC GTC Val Leu Thr Val TGG CTG Trp Leu 130 AAT GGC AAG GAG Asn Gly Lys Glu

TAC

Tyr 135 AAG TGC AAG GTC Lys Cys Lys Val AAC AAA GCC CTC Asn Lys Ala Leu

CCA

Pro 145 GCC CCC ATC GAG Ala Pro Ile Glu

AAA

Lys 150 ACC ATC TCC AAA Thr Ile Ser Lys AAA GGG CAG CCC Lys Gly Gin Pro

CGA

Arg 160 432 480 528 GAA CCA CAG GTG Glu Pro Gin Val

TAC

Tyr 165 ACC CTG CCC CCA Thr Leu Pro Pro CGG GAG GAG ATG Arg Glu Glu Met ACC AAG Thr Lys 175 -W W99/21997 AAC CAG GTC Asn Gin Val ATC GCC GTG Ile Ala Val 195 PCTIUS98/22879

AGC

Ser 180 CTG ACC TGC CTG Leu Thr Cys Leu

GTC

Val 185 AAA GGC TTC TAT Lys Gly Phe Tyr CCC AGC GAG Pro Ser Asp 190 AAC TAG AAG Asn Tyr Lys GAG TGG GAG AGC Giu Trp, Giu Ser

AAT

Asn 200 GGG CAG CCG GAG Gly Gin Pro Giu

AAC

Asn 205 ACC ACG Thr Thr 210 CCT CCC GTG CTG Pro Pro Val Leu

GAC

Asp 215 TGC GAC GGC TCC Ser Asp Gly Ser TTG CTC TAT AGC Phe Leu Tyr Ser

AAG

Lys 225 CTC ACC GTG GAG Leu Thr Val Asp AGC AGG TGG GAG Ser Arg Trp Gin GGG AAC GTC TTC Gly Asn Val Phe

TCA

Ser 240 672 720 768 TGC TGC GTG ATG Gys Ser Val Met

CAT

His 245 GAG GCT CTG CAC Giu Ala Leu His

AAC

Asn 250 CAC TAC ACG GAG His Tyr Thr Gin AAG AGC Lys Ser 255 GTC TGC CTG Leu Ser Leu TGT ACT AGT Ser Thr Ser 275

TGT

Ser 260 GGG GGT AAA GGA Pro Giy Lys Gly

GGG

Gly 265 GGG GGA TCA GGG Giy Giy Ser Gly GGC GGA GGA Gly Giy Gly 270 GAA ATA ATT Glu Ile Ile 816 864 ATC GAA TGG CAT Ile Giu Trp His

AAG

Lys 280 TTT GAA ACG AGT Phe Glu Thr Ser

GAA

Giu 285 TCT ACT Ser Thr 290 TAG TTA ATA GAT Tyr Leu Ile Asp

GAT

Asp 295 GTA TTA TAG ACG GGG GTT AAT GGG GCG Val Leu Tyr Thr Gly Vai Asn Gly Ala 912 960 GTA Val 305 TAT ACA TTT TCA AAT AAT GAA GTA AAC Tyr Thr Phe Ser Asn Asn Giu Leu Asn 310 ACT GGT TTA ACT Thr Giy Leu Thr

AAT

Asn 320 AAG AAT AAT TAT Asn Asn Asn Tyr

ATG

Ile 325 ACA ACA TGT ATA Thr Thr Ser Ile

AAA

Lys 330 GTA GAG GAT ACA Vai Giu Asp Thr TTA GTA Leu Vai 335 1008 TGG GGA ACC Cys Gly Thr

AAT

Asn 340 AAG GGA AAC CCC Asn Giy Asn Pro

AAA

Lys 345 TGT TGG AAA ATA Gys Trp Lys Ile GAG GGT TCC Asp Gly Ser 350 CAA AAT AGT Gin Asn Ser 1056 GAA GAT CCA AAA TAT AGA GGT Giu Asp Pro Lys Tyr Arg Gly 355

AGA

Arg 360 GGA TAT GCT CGT Gly Tyr Aia Pro

TAT

Tyr 365 AAA GTG Lys Val 370 ACG ATA ATG AGT Thr Ile Ile Ser

CAT

His 375 AAG GAA TGT GTA Asn Glu Cys Val

CTA

Leu 380 TCT GAT ATA AAG Ser Asp Ile Asn 1104 1152 1200 ATA Ile 385 TCA AAA GAA GGA Ser Lys Giu Gly AAA AGA TGG AGA Lys Arg Trp Arg TTT GAG GGA GCA Phe Asp Gly Pro -WO 99/21997 PCTIUS98/22879 GGT TAT GAT TTA Gly Tyr Asp Leu

TAC

Tyr 405 ACG GCA GAT AAC Thr Ala Asp Asn

GTG

Val 410 ATT CCA AAA GAT Ile Pro Lys Asp GGT GTG Gly Val 415 1248 CGT GGA GCA Arg Gly Ala

TTC

Phe 420 GTT GAT AAA GAC Val Asp Lys Asp

GGC

Gly 425 ACT TAT GAC AAA Thr Tyr Asp Lys GTT TAC ATT Val Tyr Ile 430 ATT CCG TAT Ile Pro Tyr 1296 1344 CTT TTC ACT GAT Leu Phe Thr Asp 435 ACT ATC GAC Thr Ile Asp AAG AGA ATT GTT Lys Arg Ile Val ATA GCA Ile Ala 450 CAA ATG TGC TTA Gin Met Cys Leu

AAT

Asn 455 GAC GAA GGT GGT Asp Glu Gly Gly

CCA

Pro 460 TCA TCA TTG TCT Ser Ser Leu Ser

AGT

Ser 465 CAT AGA TGG TCG His Arg Trp Ser TTT CTC AAG GTC Phe Leu Lys Val

GAA

Glu 475 TTA GAA TGT GAT Leu Glu Cys Asp 1392 1440 1488 GAC GGA AGA AGT Asp Gly Arg Ser

TAT

Tyr 485 AGA CAA ATT ATT Arg Gin Ile Ile

CAT

His 490 TCT AAA GCT ATA Ser Lys Ala Ile AAA ACA Lys Thr 495 GAT AAT GAT Asp Asn Asp TCC GCA TTA Ser Ala Leu 515

ACG

Thr 500 ATA CTA TAT GTA Ile Leu Tyr Val

TTC

Phe 505 TTT GAT AGT CCT Phe Asp Ser Pro TAT TCC AAG Tyr Ser Lys 510 1536 TGT ACC TAT TCT Cys Thr Tyr Ser AAT GCC ATT AAA CAC TCT TTT TCT Asn Ala Ile Lys His Ser Phe Ser 525 1584 ACG TCA Thr Ser 530 AAA TTG GGA GGA Lys Leu Gly Gly

TAT

Tyr 535 ACA AAG CAA TTG Thr Lys Gin Leu

CCG

Pro 540 TCT CCA GCT CCT Ser Pro Ala Pro 1632

GGT

Gly 545 ATA TGT CTA CCA Ile Cys Leu Pro

GCT

Ala 550 GGA AAA GTT GTT CCA CAT ACC ACG TTT Gly Lys Val Val Pro His Thr Thr Phe 555 1680 ATC ATA GAA CAA Ile Ile Glu Gin

TAT

Tyr 565 AAT GAG CTA GAT Asn Glu Leu Asp ATT ATA AAG CCT Ile Ile Lys Pro TTA TCT Leu Ser 575 1728 CAA CCT ATC Gin Pro Ile GAG AAG GAA Glu Lys Glu 595 GAA GGA CCG TCT Glu Gly Pro Ser

GGT

Gly 585 GTT AAA TGG TTC Val Lys Trp Phe GAT ATA AAG Asp Ile Lys 590 ATA AAA GAA Ile Lys Glu 1776 1824 AAT GAA CAT CGG Asn Glu His Arg

GAA

Glu 600 TAT AGA ATA TAC Tyr Arg Ile Tyr AAT ACT Asn Thr 610 ATA TAT TCG TTC Ile Tyr Ser Phe

GAT

Asp 615 ACA AAA TCT AAA Thr Lys Ser Lys ACT CGT AGT GCA Thr Arg Ser Ala 1872 -WO 99/21997 CAA GTT GAT GCG CGA CTA TTT TCA GTA ATG Gin Val Asp Ala Arg Leu Phe Ser Val Met 625 630 TTT ATA GCA GAT ATA GGG ATA GGA GTA GGA Phe Ile Ala Asp Ile Gly Ile Gly Val Gly 645 650 ATA CTT AAA ATG TAA Ile Leu Lys Met 660 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 661 amino acids TYPE: amino acid TOPOLOGY: linear PCT/US98/22879 GTA ACT TCG AAA CCG TTA 1920 Val Thr Ser Lys Pro Leu 635 640 ATA CCA CGA ATG AAA AAA 1968 Ile Pro Arg Met Lys Lys 655 1983 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Phe His Val Ser Phe Arg Tyr Ile Phe 1 Leu Lys Glu Asp Asp Gly Asn Trp Pro 145 Leu Ser Glu Leu Ser Glu Thr 115 Asn Pro Leu Cys Gly Met His Val 100 Tyr Gly Ile 5 Pro Asp Ala Ile Glu His Arg Lys Glu Val Lys Pro Ser 70 Asp Asn Val Glu Lys 150 Thr Thr Ser Arg Pro Ala Val Tyr 135 Thr Ser 25 Thr Phe Pro Val Thr 105 Val Cys Ser Gly Tyr Pro Phe Val 75 Phe Pro Thr Val Ala 155 Pro Asp Cys Pro Cys Trp Glu Leu 125 Asn Gly Pro Asp Pro Lys Val Tyr Glu 110 His Lys Gin Leu Asp Ala Pro Val Val Gin Gin Ala Pro Ile Asp Pro Lys Val Asp Tyr Asp Leu Arg 160 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys -:WO099/21997 PCT/US98/22879 eu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 185 190 Asn Ie Thr Lys 225 Cys Leu Ser Ser Val 305 Asn Cys Giu Lys Ile 385 Gly Arg Leu Ile Ser 465 Gin Ala Thr 210 Leu Ser Ser Thr Thr 290 Tyr Asn Gly Asp Val 370 Ser Tyr Giy Phe Aia 450 His Val Ser Li 180 Vai Giu T: 195 Pro Pro V~ Thr Vai A~ Val Met H: 2' Leu Ser P~ 260 Ser Ile G~ 275 Tyr Leu I: Thr Phe S Asn Tyr I: 3: Thr Asn A 340 Pro Lys T 355 Thr Ile I: Lys Giu G Asp Leu T Ala Phe V~ 420 Thr Asp TI 435 Gin Met C~ Arg Trp S~ s is Lu le 25 le 'is Giu Leu Lys 230 Giu Gly Trp Asp Asn 310 Thr Gly Arg Ser Ile 390 Thr Asp Ile Leu Thr 470 Ser Asp 215 Ser Ala Lys His Asp 295 Asn Thr Asn Gly His 375 Lys Ala Lys Asp Asn 455 Phe Asn 200 Ser Arg Leu Gly Lys 280 Val Giu Ser Pro Arg 360 Asn Arg Asp Asp Thr 440 Asp Leu Giy Asp Trp His Gly 265 Phe Leu Leu Ile Lys 345 Giy Giu Trp Asn Giy 425 Lys Giu Lys Gin Gly Gin Asn 250 Gly Giu Tyr Asn Lys 330 Cys Tyr Cys Arg Val1 410 Thr Arg Gly Val1 Pro Ser Gin 235 His Gly Thr Thr Lys 315 Vai Trp Ala Val1 Arg 395 Ile Tyr Ile Gly Giu 475 Asn 205 Phe Asn Thr Gly Giu 285 Val Giy Asp Ile Tyr 365 Ser Asp Lys Lys Lys 445 Ser Glu Asn Leu Val1 Gin Giy 270 Giu Asn Leu Thr Asp 350 Gin Asp Gly Asp Val 430 Ile Ser Cys Tyr Tyr Phe Lys 255 Gly Ile Giy Thr Leu 335 Giy Asn Ile Pro Giy 415 Tyr Pro Leu Asp Lys Ser Ser 240 Ser Gly Ile Aia Asn 320 Vai Ser Ser Asn Cys 400 Vai Ile Tyr Ser Ile 480 -WO 99/21997 Asp Gly Arg Asp Asn Asp Ser Ala Leu 515 Thr Ser Lys 530 Gly Ile Cys 545 Ile Ile Glu Gin Pro Ile Glu Lys Glu 595 Asn Thr Ile 610 Gin Val Asp 625 Phe Ile Ala Ile Leu Lys Ser Thr 500 Cys Leu Leu Gin Phe 580 Asn Tyr Ala Asp Met 660 Tyr 485 Ile Thr Gly Pro Tyr 565 Glu Glu Ser Arg Ile 645 Arg Gin Leu Tyr Tyr Ser Gly Tyr 535 Ala Gly 550 Asn Glu Gly Pro His Arg Phe Asp 615 Leu Phe 630 Gly Ile Ile Val Met 520 Thr Lys Leu Ser Glu 600 Thr Ser Gly Ile Phe 505 Asn Lys Val Asp Gly 585 Tyr Lys Val Val His 490 Phe Ala Gin Val Asp 570 Val Arg Ser Met Gly 650 Ser Asp Ile Leu Pro 555 Ile Lys Ile Lys Val 635 Ile Lys Ser Lys Pro 540 His Ile Trp Tyr Gin 620 Thr Pro Ala Pro His 525 Ser Thr Lys Phe Phe 605 Thr Ser Arg Ile Tyr 510 Ser Pro Thr Pro Asp 590 Ile Arg Lys Met PCT/US98/22879 Lys Thr 495 Ser Lys Phe Ser Ala Pro Phe Asp 560 Leu Ser 575 Ile Lys Lys Glu Ser Ala Pro Leu 640 Lys Lys 655 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATCGCATCAT CTACCTTCAT CCATTCCGAC CTG INFORMATION FOR SEQ ID -WO 99/21997 PCT/US98/22879 SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: primer (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID TAAACACTCC GAACAGGATT TATGTTTATT GCA 33

Claims (21)

1. An isolated VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, the binding portion being capable of binding a semaphorin.

2. An isolated VESPR polypeptide of claim 1 wherein the semaphorin to which the binding portion is capable of binding is selected from the group consisting of A39R semaphorin and AHV semaphorin.

3. An isolated VESPR polypeptide of claim 1, wherein the binding portion consists of SEQ ID NO:2.

4. The isolated VESPR (Viral Encoded Semaphorin Protein Receptor) ':.polypeptide of claim 1 consisting of an amino acid sequence that is at least identical to SEQ ID NO:2. 9 A soluble VESPR (Viral Encoded Semaphorin Protein Receptor) polypeptide comprising a binding portion consisting of an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of: 25 amino acids xl to 944 of SEQ ID NO:2, wherein xl is amino acid 1 or and a fragment of the sequence of wherein the binding portion is capable of binding a semaphorin.

6. The soluble VESPR polypeptide of claim 5 wherein the binding portion consists of amino acids x, to 944 of SEQ ID NO:2 or a fragment thereof, wherein x, is amino acid 1 or

7. An isolated polynucleotide selected from the group consisting of: a polynucleotide encoding a VESPR polypeptide comprising a binding portion consisting of an amino acid sequence at least 90% identical to SEQ ID NO:2; a polynucleotide of SEQ ID NO:1; and a polynucleotide complementary to or wherein the binding portion can bind a semaphorin.

8. The isolated polynucleotide of claim 7 wherein the binding portion consists of SEQ ID NO: 2.

9. An isolated polynucleotide selected from the group consisting of: a polynucleotide encoding a soluble VESPR polypeptide comprising a binding portion consisting of an amino acid sequence at least 90% identical to .amino acids x, to 944 of SEQ ID NO:2, wherein xl is amino acid 1 or a polynucleotide encoding a soluble VESPR polypeptide comprising a binding portion that is a fragment of the binding portion and a polynucleotide complementary to the polynucleotides of or 20 wherein the the binding portion of and/or can bind a semaphorin.

10. A polynucleotide of claim 9 wherein the binding portion consists of an amino acid sequence selected from the group consisting of: amino acids x, to 944 of SEQ ID NO:2, wherein x, is amino acid 1 or 25 and a fragment of

11. A recombinant expression vector comprising the polynucleotide of claim 7. W:/llona/Sharon/speci/sp12047 i I .'1

12. A recombinant expression vector comprising the polynucleotide of claim 9.

13. A process for preparing a VESPR polypeptide, the process comprising culturing a host cell transformed with an expression vector of claim 11 or 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 any one of claims 1-6 and 23-25. An isolated antibody that is specifically immunoreactive with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.

16. The isolated antibody of claim 15 that is specifically immunoreactive with a polypeptide consisting of amino acids 35 to 944 of SEQ ID NO:2. 20 17. Use of a polypeptide of any one of claims 1-6 and 23-25 in the manufacture of a medicament for treating an inflammatory disease in a *i mammal.

18. A method of screening for binding to a VESPR 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 of: SEQ ID NO:2; an amino acid sequence that is at least 90% identical to SEQ ID NO:2; 46 an amino acid sequence that is at least 90% identical to amino acids xl to 944 of SEQ ID NO:2 wherein x, is amino acid 1 or 35; and fragments of or wherein the binding portion is capable of binding a semaphorin.

19. The method of claim 18, wherein the binding portion consists of amino acids 35 to 944 of SEQ ID NO:2.

20. The method of claim 18, wherein the VESPR polypeptide is fused to a peptide that facilitates purification and/or identification.

21. The method of claim 18, wherein the VESPR polypeptide is bound to a solid support.

22. The method of claim 18, wherein the mixture contains cells that express a semaphorin. g 23. An isolated VESPR (Viral Encoded Semaphorin Protein Receptor) 20 polypeptide according to claim 1 or 5, substantially as hereinbefore described with reference to any one of the examples. o.

24. A fusion protein comprising a binding portion consisting of an amino acid sequence selected from the 25 group consisting of: xi to 944 where x, is amino acid 1 or 35 of SEQ ID NO:2; a fragment of and an amino acid sequence that is at least 90% identical to the amino acids of or wherein the binding portion can bind a semaphorin and another polypeptide. 47 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 a sequence selected from the group consisting of: SEQ ID NO:2; amino acids xl to 944 of SEQ ID NO:2, wherein xl is amino acid 1 or and a fragment of the sequence of or wherein the binding portion is capable of binding a semaphorin.

26. An isolated VESPR (Viral Encoded Semaphorin Protein Receptor) polynucleotide selected from the group consisting of: a polynucleotide encoding a VESPR polypeptide comprising a binding portion consisting of an amino acid sequence at least 80% identical to SEQ ID NO:2; a polynucleotide of SEQ ID NO:1; S(c) a polynucleotide encoding a VESPR polypeptide comprising a binding portion consisting of an amino acid sequence at least 80% identical to amino 20 acids x, to 944 of SEQ ID NO:2, wherein x, is amino acid 1 or 35; and a polynucleotide complementary to or wherein the binding portion of or can bind a semaphorin. DATED: 15 February 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: Immunex Corporation