MXPA97000213A - Noveled cytokine designated as ler - Google Patents

Abstract

The present invention describes LERK-5 polypeptides, together with ADM sequences, vectors and transformed host cells useful in the production of LERK-5. The polypeptides of LERK-5 bind to elk and hek, which are members of the eph / elk family of receptor tyrosine kinases.

Description

NOVELED CYTOKINE DESIGNATED AS LERK-5 BACKGROUND OF THE INVENTION Proteins known as receptor tyrosine kinases have an intrinsic kinase activity that is activated by ligand binding. This class of proteins is characterized by conserved structural motifs within the catalytic domains (Hanks et al., Science 242: 42, 1988) and can be subdivided into families based on structural features of the N-terminal regions to the catalytic domain. Boyd et al (J. Biol. Chem. 267: 3262, 1992) purified a glycoprotein from the surface of cells exhibiting tyrosine kinase activity. The N-terminal amino acid sequence identified this protein as a member of the eph / elk family, and the protein was thus designated as hek (human eph / elk type kinase). A monoclonal antibody immuno-reactive with hek was used to study the expression of hek in a number of human cell types (Boyd et al., Supra). The hek antigen was detected in the pre-B human cell leukemia cell line, LK63 (the cell line used as the immunogen against which the antibody arose) and the human T cell leukemia cell line, J M. The Raji B lymphoma cell line showed weak expression of the hek antigen, and the remaining cell lines tested (both normal and tumor cell lines, among which were hemopoietic cell lines that included pre-B lines). and T cell), were consistently negative. Of the biopsy, normal tissue and tumor specimens, which were also tested for hek antigen expression, none of the normal tissues was positive and only a very small proportion of hemoetic tumors was positive. The expression of the hek transcripts in the LK63 and JM cell lines, described above, as well as the human T cell leukemia cell line, HSB-2, has been demonstrated by Northern blot analysis. , (Wicks et al., Proc. Nati, Acad. Sci. U.SA 89: 161 1, 1992). The nucleotide and the amino acid sequences for an isolated hek cDNA clone are presented in Wicks et al., Supra. The cell surface protein designated elk is another member of the protein family of the tyrosine kinase receptor. First, a partial clone of elk was described in a cDNA expression library of a rat brain, which was selected for proteins expressing tyrosine kinase activity (Lerwin et al., Oncogene 3: 621, 19888). Next, a mixed sequence spanning the entire elk coding region was derived from partial clones from a collection of rat brain cDNA and a rat brain cerebellar collection, using the partial clone as a probe (Lhotak et al., Mol Cell Biol. 1 1: 2496, 1991). The hek and elk proteins are closely related to a number of other receptor quirosin kinases, including the homologues of hek, mek4 and cek4 (Sajjadi et al., New Biol. 3: 769, 1991); eek (Chan et al., Oncogene 6: 1057, 1991); erk (Chan et al., supra), eck (Lindberg et al., Mol. Cell, Biol. 10: 6316, 1990); cekd (Pasquale, E. B. Cell, Regulation 2: 523, 1991); and eph (Hirai et al., Science 238: 1717, 1987). The proteins of this subfamily are related not only in their cytoplasmic domains, but also in their extracellular domains, which are from 41 to 68% identical. Interestingly, the tissue distributions of these various receptors are diverse. For example, it has been reported that the expression of elk mRNA is limited to the testis and the brain (Lhotak et al., Supra), where sk was found not only in these two tissues, but also in the lung, intestine, kidney , spleen, ovaries, and skin. Those ligands that have been identified for receptor tyrosine kinases are a diverse group of proteins that affect growth, differentiation, and survival of cells that express the receptors. The ligands for hek and elk have been isolated, as discussed in more detail below. The identification of any additional ligands for hek and elk, which may exist, could prove useful in investigating the nature of cellular procedures regulated by signaling through these receptors. If it is desired to improve or inhibit a particular biological signal mediated through these receptors, it is advantageous to identify each of the proteins that can play an important role in the transduction of said signals. In addition, proteins that bind to certain receptors without initiating signal transduction are known, including the interleukin-1 receptor antagonist protein (Eisenberg et al., Nature 343: 341, 1990; Hannum et al., Nature 343: 336, 1990; and Carter et al., Nature 344: 633, 1990). The identification of any additional protein that a hek or elk, is desirable in order to determine whether such proteins function as antagonists.

COMPENDIUM OF THE INVENTION The present invention provides a novel cytokine designated LERK-5 that binds elk and a hek, which are members of the eph / elk family of receptor tyrosine kinases. The present invention also provides isolated DNA encoding the LERK-5 protein and expression vectors comprising the isolated DNA. A method for producing LERK-5 comprises culturing host cells containing the expression vectors, under conditions suitable for the expression of the LERK-5 protein, and recovering LERK-5. Antibodies directed against the LERK-5 protein are also described.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents an amino acid sequence alignment of human KER5 and LERK5 of murine LERK-5. The transmembrane region is in a box and the first amino acid of the mature protein is in a circle, for each protein. Amino acid identities are indicated by vertical lines.

DETAILED DESCRIPTION OF THE INVENTION A cDNA encoding a novel protein designated LERK-5, which binds to cell surface receptors, known as elk and hek, has been isolated according to the present invention. Expression vectors comprising LERK-5 DNA are also provided. Methods for producing recombinant LERK-5 polypeptides involve culturing host cells transformed with the expression vectors under conditions appropriate for the expression of LERK-5, and recovering the expressed LERK-5. The present invention also encompasses the purified protein of LERK-5, including soluble forms of the protein comprising the extracellular domain. The present invention also provides LERK-5 or fragments thereof which can act as immunogens to generate antibodies reactive therewith. In one embodiment, the antibodies are monoclonal antibodies specific for LERK-5.

The cDNA of human LERK-5 was successfully isolated as described in Example 1. The DNA sequence of the coding region of a human LERK-5 cDNA clone, and the amino acid sequence encoded thereby, are set forth in SEQ ID NO: 1 and SEC I D NO: 2. The encoded protein comprises an N-terminal signal peptide (amino acids-25 to -1 of SEQ ID NO: 2), an extracellular domain (amino acids 1 to 199), a transmembrane region (amino acids 200 to 225), and a cytoplasmic domain (amino acids 226 to 308). The binding of LERK-5 to the two cell surface receptors, known as elk and hek, is demonstrated in Example 4. A cell lysate, containing a recombinant phage 10 gt vector comprising LERK-5 cDNA, It was deposited at the American Type Culture Collection on June 16, 1994 and assigned access No. ATCC 75815. The deposit was made under the terms of the Budapest Treaty. The recombinant gt 10 vector was that isolated from Example 1 and identified as clone 6. The novel cytokine described herein, is a ligand for elk, a rat cell surface receptor that is a member of the eph / elk family of receptor tyrosine kinases. The expression of elk mRNA has been detected in the brain and rat test (Lhotok et al., Supra), and the possibility that elk is capable of oncogenic activation has been suggested (Letwin et al., Supra). The cytokine is also a ligand for hek, which is another member of the eph / elk family of receptor tyrosine kinases (Boyd et al., J. Biol. Chem. 267: 3262, 1992; Wicks et al., PNAS U SA 89: 161 1, 1992). Among cell types in which hek is expressed, certain lines of human leukemia / -v cells are found. The term "LERK-5P" as used herein, refers to a genus of polypeptides, which are capable of binding elk and hek, and exhibits substantial homology to the amino acid sequences of LERK-5, described here. Human LERK-5 is within the scope of the present invention, as are LERK-5 proteins derived from other mammalian species, including, but not limited to, murine, rat, bovine, porcine, or several primate species. As used herein, the term "LERK-5" includes membrane binding proteins (comprising an extracellular domain, a transmembrane region, and a cytoplasmic domain), as well as truncated proteins that retain the property of elk binding or hek binding. Such truncated proteins include, for example, soluble LERK-5, which comprises only the extracellular domain (receptor binding). Other proteins that bind elk and hek have been identified. The LERK-5 of the present invention is a novel protein, distinct from the other elk binding and hek binding proteins. An elk ligand is described in the PCT application WO 94/1 1384. The nucleotide sequence of the cloned cDNA (designated clone tele7) encoding this elk ligand, as well as the amino acid sequence encoded therein, are presented in WO 94/1 138. The protein is a protein of transmembrane type I, comprising 346 amino acids, including an N-terminal signal peptide, a extracellular domain, a transmembrane domain, and a cytoplasmic domain. In the PCT application WO 95/06065, two hek ligand proteins (encoded by clones designated AD and C6) are described, which are 38% identical at the amino acid level. The DNA and amino acid sequences encoded for the cloned cDNA, which encodes the two hek ligand proteins, are presented in the application WO 95/06065. The encoded proteins, each containing an N-terminal signal peptide, an extracellular domain, and a C-terminal hydrophobic region containing signals for the anchoring of glycophosphatidylinositol (GPI). After post-translational processing, both proteins are anchored to the cell surface via G PI binding. Another protein that has been found to bind elk and hek is designated B61. The nucleotide and the amino acid sequences encoded for B61 cDNA are presented in Holzman et al. (Mol, Cell, Biol. 10: 5830, 1990). Subsequently, B61 was found to bind elk and hek, as demonstrated in the binding analyzes described in WO 95/06065. Of these four proteins (B61 and the proteins encoded by the tele 7, A2, and C6 clones), the LERK-5 of the present invention is the one most closely (structurally) related to the elk ligand encoded by tele 7 (designated as elk). -Left 7, from here on out). The amino acid sequences of the LER K-5 and elk-L tele 7 proteins, of full length, are 58.5% identical, and the DNA sequences are 61.4% identical. The sequences comprising the signal peptide and the extracellular domain of elk-L and LERK-5 are 51.6% identical at the amino acid level, and 53.6% identical at the DNA level. The cytoplasmic domains are 74.7% identical at the amino acid level, and 75.5% identical at the DNA level. Another member of the receptor tyrosine kinase family is hek2 (Bóhme et al., Oncogene 8: 2857, 1993). LERK-5 joins hek2. The nucleotide sequence of a human expressed sequence tag (EST) for chromosome 13 was found in GenBank® under accession number L13819. The source of the EST is identified as the cDNA derived from mRNA from the brain of a 3-month-old human female. When the 337bp sequence described for the EST is aligned with the elk-L tele 7 nucleotide sequence, the sequence tag is 59.3% identical to the corresponding region of the elk-L tele 7 DNA. In view of this degree of homology of sequence, the inventors of the present seek to determine whether or not the sequence tag formed part of a gene encoding a biologically active protein, specifically, a protein that could bind elk or hek. The GenBank registry does not disclose any polypeptide encoded by EST (e.g., it does not indicate what the reading frame may be, if it exists), much less suggests that any polypeptide could bind elk or hek. Even if the sequence tag A DN was expressed in the reading frame elucidated by the cloning of the LERK-5 DNA reported herein, the amino acid sequence, ----- • encoded might lack three or more of the four cysteine residues that are conserved in the extracellular domains of the four proteins described above, which bind elk and hek. In the LERK-5 protein of the present invention, these cysteines are found in amino acids 37, 64, 76, and 128 of SEC I D NO: 2. A translation of the sequence tag could correspond to amino acids 79 to 190 of SEQ ID NO: 2 (with a mismatch, described in Example 1). In this manner, the 337bp sequence tag is not thought to encode a polypeptide capable of binding elk or hek. The human LERK-5 cDNA, isolated in Example 1 below, can be radiolabeled and used as a probe to isolate other mammalian LERK-5 cDNAs by cross-hybridization of species. RNAs isolated from several cell lines or tissues derived from different mammalian species may be selected by Northern hybridization to identify a suitable source of mRNA to be used in the cloning of a LERK-5 gene. The DNA encoding murine LERK-5 was isolated from a collection of cDNA prepared from 1.5-day embryos. The The amino acid sequence of murine LERK-5 is presented, and is aligned with the amino acid sequence of human LERK-5, in Figure 1. The LER K-5 of murine and human are 97% identical to the amino acid level. The fragments of the LERK-5 protein of SEC I D NO: 2, which are capable of uNir elk or hek are encompassed by the present invention. One embodiment of the present invention provides soluble LERK-5 polypeptides. The soluble LERK-5 polypeptides comprise all or part of the extracellular domain of a native KER-5 LER, but lack the transmembrane region that could cause retention of the polypeptide on a cell membrane. The soluble LERK-5 polypeptides advantageously comprise the native signal peptide (or a heterologous), when they are initially synthesized to promote secretion, but the signal peptide is separated by the secretion of LERK-5 from the cell. The soluble LERK-5 polypeptides that can be employed retain the ability to bind elk or hek. Soluble LERK-5 may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble LERK-5 protein is capable of being secreted. Soluble LERK-5 can be identified (and distinguished from its non-soluble membrane-binding counterparts) by separating the intact cells, which express the desired protein, from the culture medium, e.g. , by centrifugation, and analyzing the medium (supernatant) for the presence of the desired protein. The presence of LERK-5 in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein. Soluble LERK-5 can be a natural form of this protein. The use of soluble forms of LERK-5 is advantageous for certain applications. Purification of the proteins from recombinant host cells is facilitated, since soluble proteins are secreted from the cells. In addition, soluble proteins are generally suitable for intravenous administration. Examples of soluble LERK-5 polypeptides include those that comprise the entire extracellular domain of a native LERK-5 protein. One of said soluble LERK-5 protein comprises amino acids 1 to 199 of SEQ ID NO: 2. When initially expressed within a host cell, the soluble protein may further comprise one of the heterologous signal peptides, described below, which is functional within the host cells employed. Alternatively, the protein may comprise the native signal peptide, such that LERK-5 comprises amino acids 25 to 199 of SEC I D NO: 2. The DNA sequences encoding soluble LERK-5 proteins are encompassed by the present invention. Truncated LERK-5, including soluble polypeptides, can be prepared by any of the conventional techniques. A desired sequence of DNA can be chemically synthesized using known techniques. It can also be produced DNA fragments by restriction endonuclease digestion of a full-length cloned DNA sequence and isolated by electrophoresis on agarose gels. Oligonucleotides that reconstruct the 5 'or 3' end of a DNA fragment can be synthesized at a desired point. The oligonucleotide can containing a restriction endonuclease cleavage site, towards the 5 'end of the desired coding sequence, and the position of an initiation codon (ATG) at the 5' end of the coding sequence. Linkers containing restriction endonuclease cleavage sites can be used to insert the desired fragment of DNA into an expression vector. The well-known polymerase chain reaction method can also be employed to isolate a DNA sequence encoding a desired protein fragment. The oligonucleotides that define the terms of the desired fragment are used as primers in PCR. As a further alternative, known mutagenesis techniques can be employed to insert a stop codon at a desired point, e.g. , immediately towards the 3 'end of the codon for the last amino acid of the extracellular domain. With respect to the above discussion of the signal peptides and the various domains of the LERK-5 protein, those skilled in the art will recognize that the limits described above for said regions of the protein are approximate. For example, although computer programs are available that predict on site of signal peptide cleavage, the cleavage may occur at sites other than those pre-stipulated. Furthermore, it is recognized that a protein preparation can comprise a mixture of protein molecules having different N-terminal amino acids, due to the cleavage of the signal peptide at more than one site. In addition, the exact boundaries of a transmembrane region may differ from those preset by a computer program. Post-translational processing, which may vary according to the particular expression system employed, may produce proteins that have N- or C-terminals that differ from those described above. Said variants that retain the desired biological activity are included among the LERK-5 polypeptides of the present invention. The present invention provides purified LERK-5 polypeptides, both recombinant and non-recombinant. Also within the scope of the present invention are variants and derivatives of native LERK-5 proteins that retain the desired biological activity (e.g., the ability to bind elk or hek). Variants of LERK-5 can be obtained by mutations of nucleotide sequences encoding native LERK-5 polypeptides. A variant of LERK-5, as it is referred to herein, is a polypeptide substantially homologous to a native LERK-5, but which has an amino acid sequence different from that of a native LERK-5, due to one or more deletions , insertions or substitutions. A variant nucleotide or amino acid sequence is preferably at least 80% identical to a native LERK-5 sequence, most preferably at least 90% identical. In one embodiment of the present invention, a LERK-5 protein comprises an extracellular domain, a transmembrane region, and a cytoplasmic domain, wherein the amino acid sequence of said LER K-5 protein is at least 90% identical to the sequence presented as amino acids 1 to 308 of S EC ID NO: 2. In another embodiment, a soluble LER K-5 polypeptide, capable of binding elk and hek, comprises an amino acid sequence that is at least 90% identical to the sequence presented as amino acids 1 to 199 of SEC I D NO: 2. The percentage of identity can be determined, for example, by comparing the sequence information using the GAP computer program, version 6.0, described by Devereux et al. (Nucí Acids, Res 12: 387, 1984), and available from the University. of U niversity of Wisconsin Genetics Computer Group (UWGCG). The GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970), and reviewed by Smith and Waterman (Adv. Appl. Math. 2: 482, 1981). Preferred error parameters for the GAP program include: (1) a unit comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the heavy comparison matrix of Gribskov and Burgess, Nucí. 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; (2) a penalty of 3.0 for each hole and an additional penalty of 0.10 for each symbol in each hole; and (3) no penalty for extreme gaps. Alterations of the native sequence can be obtained by any number of known techniques. Mutations can be introduced into particular foci, synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting, reconstructed sequence encodes an analog having the desired insertion, substitution or elimination of the amino acid. Alternatively, site-specific mutagenesis methods directed to the oligonucleotide can be employed to provide an altered gene having particular codons altered in accordance with the substitution, deletion or insertion required. Examples of methods for making the alterations, previously established, are described 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. Nati. Acad. Sci. U.S.A. 82: 488, 1985); Kunkel et al (Methods in Enzymol., 154: 367, 1987); and E patents. U .A. Nos. 4, 518, 584 and 4,737,462, which are incorporated herein by reference. The variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Me, Val, Leu, or Ala, for another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other conservative substitutions are also known, for example, substitutions of whole regions having similar characteristics of hydrophobic character. LERK-5 can also be modified to create derivatives of LERK-5 by forming covalent conjugates or aggregates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, and the like. The covalent derivatives of LERK-5 can be prepared by linking the chemical moieties to functional groups on amino acid side chains of LERK-5 or on the N-terminus or C-Term of a LERK-5 polypeptide, or its extracellular domain. Other derivatives of LERK-5, within the scope of this invention, include covalent or aggregated conjugates of LERK-5 or its fragments with other proteins or polypeptides, such as by synthesis in the recombinant culture as N-terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence (e.g., the leader of Saccharomyces factor a) in the N-terminus of a LERK-5 polypeptide. The signal peptide or co-translational leader or post-translationally directs the transfer of the conjugate from its synthesis site to a site inside or outside the membrane of the cell or the cell wall. LERK-5 peptide fusions may comprise peptides added to facilitate the purification and identification of LER K-5. Such peptides include, for example, poly-His or the antigenic identification peptides described in the patent of E.U.A. Do not . 5.01 1, 912 (incorporated herein by reference, and in H opp et al., Bio / Technology 6: 1204, 1988. One of said peptides is the peptide FLAG®, Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys (DYKDDDDK) (SEQ ID NO: 3), which is highly antigenic and provides a reversibly linked epitope by a specific monoclonal antibody that allows the rapid analysis and easy purification of the expressed recombinant protein.This sequence is also specifically separated by the enterokinase of the bovine mucosa in the residue immediately after the Asp-Lys pair The present invention also includes LERK-5 polypeptides, with or without glycosylation of the native model, associated. LERK-5 expressed in yeast or mammalian expression systems (eg, COS-7 cells) may be similar to or significantly different from a native LERK-5 polypeptide in a molecular weight and glycosylation pattern, depending on the choice of expression system. The expression of LERK-5 polypeptides, in bacterial expression systems, such as E. coli, provides molecules without glycosylation. The N-glycosylation sites in the extracellular domain of LERK-5 can be modified to prevent glycosylation. Such sites in eukaryotic peptides 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 human LERK-5 protein comprises two such triplets, at amino acids 1 1 - 13 and 1 14-1 16 of SEC I D NO: 2. Appropriate modifications to the nucleotide sequence, which encodes this triplet, will result in substitutions, additions or deletions, which prevent binding of carbohydrate residues to the Asn side chain. The alteration of an individual 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 the N-glycosylation sites in proteins include those described in the U.S.A. patent. 5,071, 972 and EP 276,846, incorporated herein by reference. In another example, the sequences encoding Cys residues, which are not essential for biological activity, can be altered to cause the Cys residues to be eliminated or replaced with other amino acids, preventing the formation of disulfide bridges, intramolecular, incorrect , after resaturation. Other variants are prepared by modifying adjacent dibasic amino acid residues to improve expression in yeast systems, in which the activity of the KEX2 protease is present. Ep 212,914 describes the use of site-specific mutagenesis to inactivate the KEX2 protease processing sites in a protein. The KEX2 protease processing sites are inactivated by elimination, addition or substitution residues to alter the pairs of Arg-Arg, Arg-Lys, and Lys Arg, to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairs are considerably less susceptible to KEX2 cleavage, and the conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred aspect for inactivating KEX2 sites. Human LERK-5 contains four KEX2 protease processing sites, at amino acids 228-229, 229-230, 232-233, and 251-252 of SEC I D NO: 2. The present invention also encompasses LER K-5 variants of natural existence. Examples of such variants are proteins that result from binding, alternative, mRNA, or proteolytic cleavage of the LERK-5 protein, wherein the elk binding or hek binding property is retained. The alternative binding of mRNA can produce a truncated, but biologically active LERK-5 protein, such as, for example, a soluble form of natural existence, of the protein. For example, variations attributable to proteolysis include differences in terms N- or C- by expression in different types of host cells, due to the proteolytic removal of one or more terminal amino acids from the LERK-5 protein ( generally of 1-5 strict amino acids). Variants that have the ability to join elk or hek, can be identified by any suitable analysis. For example, the biological activity of a variant of LERK-5 can be determined by analyzing the ability of the variant to compete with LERK-5 to bind elk or hek (ie, in competition binding analysis). Combat binding analyzes can be performed using normal techniques. For example, radiolabelled LERK-5 can be used to compete with a variant of LERK-5 to analyze the binding to the cell surface-bound to elk or hek. Qualitative results can be obtained by competitive autoradiographic plate binding analysis, or Scatchard graphs can be used to generate quantitative results. Instead of intact cells, the binding of soluble elk or hek can be substituted to a solid phase (eg, a soluble fusion protein of elk / Fc or hek / Fc bound to a solid phase containing Protein A or Protein). G). Another type of competitive binding analysis uses elk or soluble hek radiolabel, and intact cells that express LERK-5. The LERK-5 of the present invention can also be used in a binding assay to detect cells expressing elk or hek. For example, LERK-5 or its extracellular domain can be conjugated to a detectable portion, such as a radionuclide, an enzyme that can catalyze a colorometric or fluorometric reaction, biotin or avidin. The cells to be tested for the expression of elk or hek are in contact with the labeled LERK-5. After incubation, labeled LERK-5 is separated from the cells, and the binding is measured using the detectable portion. One aspect of the present invention involves the use of LERK-5 to bind elk or hek proteins. For example, LERK-5 can be used as a reagent in protein purification procedures. The LERK-5 or LERK-5 / Fc fusion proteins are linked to a solid support material by conventional techniques, and used to purify elk or hek by affinity chromatography. The LERK-5 proteins described herein can also be used to measure the biological activity of the elk or hek proteins, in terms of their binding affinity for LERK-5. As an example, LERK-5 can be used to determine if biological activity is retained after modification of an elk or hek protein (e.g., chemical modification, truncation, mutation, etc.). The biological activity of an elk or hek protein in this way can be ascertained before it is used in a research study, for example. The LERK-5 proteins find use as reagents that can be used by those conducting "quality assurance" studies eg to verify the storage life and stability of the elk protein under different conditions. 5 can be used in a binding affinity study to measure the biological activity of an elk protein that has been stored at different temperatures, or produced in different cell types.The binding affinity of the modified elk protein for LERK-5 is It compares with that of an unmodified elk protein to detect any adverse impact of changes in the biological activity of elk.Also, the biological activity of a hek protein can be analyzed using LERK-5 .The polypeptides of LERK-5 also find use as carriers for delivering agents bound thereto to cells that carry the elk or hek cell surface receptor. The expression of the hek antigen for certain leukemic cell lines, including the human T cell leukemia cell line designated JM, and the human pre-B cell leukemia cell line designated as LK63 (Boyd et al. J. Biol. Chem. 267: 3262, 1992, and Wicks et al., Proc. Nati Acad. Sci. U.S. A. 89: 1611, 1992). In this way, LERK-5 proteins can be used to deliver diagnostic or therapeutic agents to these cells (or to other cell types found to express hek on the cell surface), in in vitro or in vivo procedures. An example of such use is to expose a hek + leukemic cell line to a therapeutic agent / LERK-5 conjugate, to analyze whether the agent exhibits cytotoxicity towards the leukemia cells. A number of different therapeutic agents can be included, linked to LERK-5, in an analysis to detect and compare the cytotoxic effect of agents on leukemia cells. Conjugates of LERK-5 / diagnostic agent can be used to detect the presence of hek + cells in vitro or in vivo. Diagnostic and therapeutic agents that can be linked to a polypeptide of LERK-5 include, but are not limited to drugs, toxins, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorimetric reaction, and the like, the particular agent being chosen in accordance with the intended application.

- Examples of drugs include those used in the treatment of various forms of cancer, eg. , nitrogen mustard, such as nitrogen mustard of L-phenylalanine or cyclophosphamide, intercalating agents such as cis-diaminodichloroplatin, antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine, and antibiotics, such as bleomycin, doxorubisin, daunorubisin, and their derivatives. Among the toxins are ricin, abrin, dipteria toxin, Apseudomonas aeruginosa exotoxin, ribosomal inactivation proteins, mycotoxins such as trichothecenes, and their derivatives and fragments (eg, individual chains). Suitable radionuclides for diagnostic use include, but are not limited to, 123 l, 131 l, 99mTc, 1 l 1 ln, and 76 Br. Suitable radionuclides for therapeutic use include, but are not limited to, 131 l, 21 1At, 77 Br, 186 Re, 18βRe, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu. Such agents can be attached to the LER K-5 by any conventional suitable method. LERK-5, being a protein, comprises functional groups on the amino acid side chains that can react with functional groups on a desired agent to form, for example, covalent bonds.

Alternatively, the protein or agent can be derivatized to generate or join a reactive, desired functional group. Derivatization may involve the joining of one of the bifunctional coupling reagents, available for the attachment of several molecules to proteins (Pierce Chemical Company, Rockford, Illinois).

A number of techniques for radiolabelling proteins are known. The radionuclide metals can be linked to LERK-5 using, for example, a suitable bifunctional chelating agent. In this manner, conjugates comprising LERK-5 and a suitable diagnostic or therapeutic agent (preferable and covalently crosslinked) are prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application. Another use of LERK-5 of the present invention is as a research tool to study the role that LERK-5, together with elk or hek, can play in the growth or differentiation of cells carrying the elk or hek receptor. The LERK-5 polypeptides of the present invention can also be used in in vitro analysis, for the detection of elk or LERK-5 or their interactions. Likewise, LERK-5 finds use in analysis for hek or the interaction of LERK-5 with hek. As discussed above, when several rat tissues were analyzed for elk mRNA, transcripts were detected only in the brain and testis (Lhotak et al., Supra). The binding of LERK-5 to elk, in neural tissue, is believed to exert a neuroprotective or neurotrophic effect. LER K-5 finds use as a tissue culture reagent. A protein of LERK-5 can be added to neurons cultured in vitro to improve the viability or prolong the life of cultured neurons, thus facilitating neural tissue research studies. The appropriate concentrations of LERK-5 can be determined by routine testing to be added to the culture medium. One embodiment of the present invention is directed to a method for treating neural tissue disorders, which involves contacting the neural tissue with LERK-5. Such disorders include bath or neurological diseases, either chronic or acute. A LERK-5 protein can be administered to a mammal to treat said damage or disease. In one embodiment of the invention, LERK-5 is used to treat neurodegenerative conditions characterized or mediated, at least in part, by the mechanism of neural death, known as excitotoxicity. In addition, LERK-5 can be administered to a mammal to exert a trophic effect on the neural tissue. In a patient suffering from loss of or damage to neurons due to injury or disease, LERK-5 can improve the viability of those neurons that have survived. The present invention provides pharmaceutical compositions comprising an effective amount of a purified LERK-5 polypeptide and a diluent, excipient, or suitable vehicle. These vehicles will be non-toxic to patients, at the doses and concentrations used. Ordinarily, the preparation of said compositions details the combination of a mammalian LER K-5 polypeptide or derivative thereof, with pH regulators, antioxidants such as ascorbic acid, low molecular weight peptides (less than about 10 residues), proteins , amino acids, carbohydrates including glucose, sucrose, or dextran, chelating agents such as E DTA, glutathione, or other stabilizers or excipients. An appropriate diluent is neutral pH regulated saline. For therapeutic use, the compositions are administered in a form and dosage appropriate to the indication and to the patient. As will be understood by those skilled in the art, a therapeutically effective dose will vary in accordance with such factors, as well as the nature and severity of the condition to be treated, and the age, size, and condition of the patient. Administration can be by any suitable route, including, but not limited to, continuous infusion, local infusion during surgery, intraventricular infusion (which may involve the use of an intraventricular catheter), sustained release of implants (gels, membranes, and similar), or injection (eg, injection at the site of injury or injection into the central nervous system.) The compositions of the present invention may contain a LERK-5 protein in any form described above, including variants thereof, biologically active derivatives and fragments In one embodiment of the invention, the composition comprises a human soluble LERK-5 protein Said protein may comprise the extracellular domain of human LERK-5 fused to a Fe polypeptide, as described above.

Oligomeric Forms of LERK-5 The present invention encompasses LERK-5 polypeptides in the form of oligomers, such as dimers or trimers. Said oligomers may be naturally occurring or produced by recombinant DNA technology. The oligomers may comprise polypeptides of LERK-5 (preferably the extracellular domain or a fragment thereof), linked by disulfide bonds or expressed as a fusion protein with or without peptide linker spacer. Oligomers can be formed by disulfide bonds between cysteine residues on different polypeptides of LERK-5, for example. The LER K-5 oligomers can be prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins, comprising heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fe domain) has been described, for example, by Ashkenazi et al. (PNAS U. SA 88: 10535, 1991) and Byrn et al. (Nature 344: 677, 1990), incorporated herein by reference. In one embodiment of the invention, a LERK-5 dimer is created by fusing LERK-5 to the Fe region of an antibody (IgG 1). The Fe polypeptide is preferably fused to the C terminus of a soluble LERK-5 (comprising only the extracellular domain). A gene fusion encoding LERK-5 / Fe fusion protein is inserted into an appropriate expression vector. The LERK-5 / Fe fusion protein is expressed in host cells transformed with the recombinant expression vector, and can be very similar to the antibody molecules, whereby disulfide bonds are formed between chains, between the Fe polypeptides, producing divalent LERK-5. Fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an oligomer of LERK-5 with four extracellular regions of LERK-5. The term "Fe polypeptide", as used herein, includes native and mutein forms of polypeptides derived from the Fe region of an antibody. Also included are truncated forms of said polypeptides, containing the region of articulation that promotes dimerization. A suitable Fe polypeptide, described in the PCT application WO 93/10151, is a single chain polypeptide extending from the N-terminal articulation region to the native C-terminus. A mutein of this Fe polypeptide is described in Example 3, below. The mutein exhibits reduced affinity for Fe receptors. Alternatively, polypeptides of LERK-5 (preferably two soluble LERK-5 polypeptides) can be linked via a peptide linker. Peptide linkers, suitable for joining polypeptides, are known and can be employed by conventional techniques. Fusion proteins, comprising LERK-5 polypeptides linked by peptide linkers, can be produced, for example, by recombinant A DN technology. The present invention provides oligomers of LERK-5, its extracellular domains or fragments, linked by disulfide interactions, or expressed as fusion polymers with or without amino acid linking groups, spacer. For example, a dimer of the extracellular domain of LERK-5 can be linked by a linking group of IgG Fe region.

Expression Systems The present invention provides recombinant expression vectors for the expression of LERK-5 and host cells transformed with the expression vectors. Any suitable expression system can be employed. The vectors include a DNA sequence of LERK-5 operably linked to regulatory, transcriptional, or translational nucleotide sequences, such as those derived from a mammalian, microbe, viral or insect gene. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, a ribosomal binding site of m RNA, and appropriate sequences that control initiation and the term transcription and translation. The nucleotide sequences are operably linked when the regulatory sequence functionally refers to the DNA sequence of LERK-5. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence of LERK-5, if the promoter nucleotide sequence controls the transcription of the LERK-5 DNA sequence. The ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants can be further incorporated into the expression vector are identified.

In addition, sequences encoding appropriate signal peptides that are not native to the LERK-5 gene can be incorporated into the expression vectors. For example, a DNA sequence, for a signal peptide (secretory leader), can be fused in frame to the sequence of LER K-5, so that LERK-5 is initially translated as a fusion protein comprising a signal peptide. A signal peptide, which is functional in the intended host cells, enhances the extracellular secretion of the LER K-5 polypeptide. The signal peptide is separated from the LERK-5 polypeptide by the secretion of LERK-5 from the cell. Suitable host cells for the expression of LERK-5 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Cloning and expression vectors suitable for use with cellular hosts of bacteria, fungi, yeast and mammals are described, for example, by Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems can also be employed to produce LE R K-5 polypeptides using AR Ns derived from DNA constructs, described herein. Prokaryotes include gram negative or g ram positive organisms, for example E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and several other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, an LER K-5 polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met can be separated from the recombinant LERK-5 polypeptide, expressed. Expression vectors for use in prokaryotic host cells generally comprise one or more selectable, phenotypic marker genes. A selectable, phenotypic marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or that provides an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids, such as the cloning vector pBR322 (ATCC 37017). PBR322 contains genes for ampicillin and tetracycline resistance, and thus provides simple means to identify transformed cells. An appropriate promoter and a DNA sequence of LERK-5 are inserted into the vector pBR322. Other commercially available vectors include, for example, pKK223-2 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wl, E.U.A.). Promoter sequences commonly used for prokaryotic host cell expression vectors include β-lactamase (penicillinase), lacrosa promoter system (Chang et al, Nature 275: 615, 1978, and Goeddel et al., Nature 281: 544, 1979) , tryptophan (trp) promoter system (Goeddel et al., Nucí Acids Res. 8: 4057, 1980; and EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage promoter? P and a thermolabile repressor sequence. Plasmid vectors, available from the American Type Culture Collection, which incorporate derivatives of the promoter? PL, include the plasmid pH UB2 (resident in the strain of E. coli, JMB9 (ATCC 37092)) and pPLc28 (resident in the strain of E. coli RR 1 (ATCC 53082)). LERK-5 can alternatively be expressed in yeast host cells, preferably of the genus Sccharomyces (e.g., S. cerevisiae). Other genera of the yeast, such as Pichia or Kluyveromyces, can also be used. Yeast vectors will usually contain a replication sequence origin of a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable labeled gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate 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 and others, Biochem. 17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase . Other vectors and promoters suitable for use in the expression of yeast are described in Hitzeman, EPA-73,657. Another alternative is the glucose-responsive ADH2 promoter, described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Connection vectors replicable in both yeast and E. coli can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp 'gene and origin of replication) to the yeast vectors described above. The leader sequence of yeast factor a can be used to direct the secretion of the LERK-5 polypeptide. The leader sequence of factor a sequence is usually inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al., Cell 30: 933, 1982; Bitter et al., Proc. Nati Acad. Sci. U .S. A. 81: 5330, 1984; E patent U .A. 4, 546.082; and EP 324,274. Other leader sequences suitable for facilitating the secretion of recombinant polypeptides from yeast hosts are well known to those skilled in the art. A leader sequence can be modified near its 3 'end to concentrate one or more restriction sites. This will facilitate the fusion of the leader sequence to the structural gene. Yeast transformation protocols are known to those skilled in the art. A protocol is described by Hinnen et al., Proc. Nati Acad. Sci. OR . S. A. 75: 1929, 1978. The Hinnen et al. Protocol selects for Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg / ml of adenine and 20 μg / ml of uracil. Yeast host cells, transformed by vectors containing the ADH2 promoter sequence, can be grown to induce expression in a rich medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% of glucose supplemented with 80 μg / ml of adenine and 80 μg / ml of uracil.Protection of the ADH2 promoter occurs when glucose leaves the medium.You can also use mammalian or insect host cell culture systems. for expressing the recombinant LERK-5 polypeptides Baculovirus systems for the production of heterologous proteins in insect cells have been reviewed by Luckow and Summers, Bio / Technology 6: 47 (1988). of mammalian origin Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651), (Gluzman et al., Cell 23: 17 5, 1981), L cells, 293 cells, C 127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary cells (CHO), HeLa cells, and BH K cell lines (ATCC CRL 10), and the cell line CV-1 / EBNA-1 derived from the African green monkey kidney cell line (ATCC CCL 70), as described by McMahan et al. (EMBO J. 1 0: 2821, 1 991).

Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Promoter sequences and enhancer sequences, commonly used, are derived from Polyoma virus, Adenovirus 2, Simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, of SV40 origin, first and last promoter, enhancer, binding and polyadenylation sites, can be used to provide other genetic elements for the expression of a structural gene sequence in a cell mammalian host. The first and last viral promoters are particularly useful, since both are readily obtained from a viral genome as a fragment, which may also contain a viral origin for replication (Fiers et al., Nature 273: 13, 1978). Larger or smaller SV40 fragments, which provide the approximately 250 bp sequence extending from the Hind III site to the Bgl I site located in the SV40 viral origin of the replication site, may also be used. Exemplary expression vectors for use in mammalian host cells can be constructed as described by Okayama and Berg (Mol, Cell, Biol. 3: 280, 1983). A useful system for high stable level expression of mammalian DNAs in murine C 127 murine epithelial cells can be constructed substantially as described by Cosman et al. (Mol Immunol., 23: 935, 1986). A highly useful 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 . Other suitable vectors can be derived from retroviruses. Instead of a native signal sequence, a heterologous signal sequence can be added, such as the signal sequence for interleukin-7 (I L-7), described in the patent of E. U.A. 4, 965, 195; the signal sequence for the interleukin-2 receptor, described in Cosman et al., Nature 312: 768 (1984); the interleukin-4 signal peptide, described in EP 367,566; the signal peptide of interleukin-1 receptor type I, described in the patent of E. U.A. 4,968,607; and the signal peptide of interleukin-1 receptor type I I, described in EP 460,846.

LERK-5 Protein The present invention provides the purified LERK-5 protein, which can be produced by recombinant expression systems, as described above, or purified from naturally occurring cells. Advantageously, the LER K-5 is purified so that no protein band, corresponding to other proteins, is detectable after analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be appreciated by one skilled in the art that multiple bands corresponding to the LERK-5 protein can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like, as discussed above. The LERK-5 protein is considered purified provided that no bands corresponding to different proteins are visualized (without LERK-5). LERK-5 is most preferably purified to substantial homogeneity, as indicated by an individual protein band after analysis by SDS-PAG E. A method for producing the LERK-5 protein comprises culturing a host cell transformed with a vector of expression comprising a DNA sequence encoding LERK-5 under conditions such that LERK-5 is expressed. Then, the LERK-5 protein is recovered from the culture medium or cell extracts, depending on the expression system used. As will be recognized by one skilled in the art, methods for purifying recombinant LERK-5 will vary according to such factors as the type of host cells employed and whether or not LERK-5 is secreted into the culture medium. For example, when expression systems that secrete the recombinant protein are employed, the culture medium can first be concentrated, using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a purification matrix, such as a gel filtration medium. Alternatively, an anion exchange resin, for example, a matrix or substrate having diethylaminoethyl pendant groups (DEAE) may be employed. The matrices that can be commonly used in protein purification are acrylamide, agarose, dextran, cellulose or other types. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include several insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more inverted phase high performance liquid chromatography (RP-HPLC) steps may be employed, employing hydrophobic RP-HPLC media, (e.g., silica gel having aliphatic methyl pendant groups or others), to further purify LERK-5. Some or all of the above purification steps, in various combinations, can be employed to improve a substantially homogeneous recombinant protein. It is also possible to use an affinity column comprising the ligand binding domain of elk or hek to LERK-5 polypeptides expressed by affinity-purity. The LERK-5 polypeptides can be recovered from an affinity column in an elution pH regulator with a high salt content and then dialysed to a pH regulator with a lower salt content, for use. Alternatively, an immunoaffinity column may comprise an antibody that binds LERK-5. Some proteins of LERK-5 / Fe fusion can be purified using a chromatography matrix having Protein A or Protein G attached thereto. The recombinant protein, produced in a bacterial culture, is usually isolated by initial disruption of host cells, centrifugation, extraction of cell pellets, and whether it is an insoluble polypeptide, or from the supernatant fluid if it is a soluble polypeptide, followed by one or more steps of concentration, salting, ion exchange, affinity purification or size exclusion chromatography. Finally, R P-H PLC can be used for the final purification steps. The microbial cells can be separated by any convenient method, including cyclic freeze-thaw breaking, sound application, mechanical, or cell lysing agents. In transformed yeast host cells, LERK-5 is preferably expressed as a secreted polypeptide to simplify purification. The secreted recombinant polypeptide can be purified by methods analogous to those described by Urdal et al. (J. Chromatog, 296: 171, 1984). U rdal et al. Describe a method including two sequential steps, of reverse phase H PLC, for the purification of recombinant human I L-2 on a preparative H PLC column.

Nucleic Acids The present invention also provides nucleotide sequences of LER K-5. Said nucleotide sequences include, but are not limited to, the LERK-5 DNA described herein, in an individual chain form and in a double-chain form, as well as its RNA complement. The LERK-5 DNA of the present invention includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA can be isolated by conventional techniques using the cDNA isolated in Example 1, or a suitable fragment thereof, as a probe. Examples of LERK-5 DNAs of the present invention include, but are not limited to, DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 1 to 1002 of SEQ ID NO: 1 (encoding the entire length of LERK-5 of SEC IDIN: 2); nucleotides 76 to 1002 of SEC I D NO: 1 (encoding the entire length of mature LERK-5); nucleotides 1 to 672 of SEC I D NO: 1 (coding for the signal peptide and the extracellular domain); and nucleotides 76 to 672 of SEQ ID NO: 1) encoding the extracellular domain). Due to the known degeneracy of the genetic code, more than one codon can encode the same amino acid. In this way, a DNA sequence can vary from those described above, yet it encodes a polypeptide having the same amino acid sequence. Said variant DNA sequences may result from silent mutations, e.g. , which may occur during PCR amplification. Alternatively, said silent mutations that are degraded as a result of the genetic code to a DNA sequence encoding LERK-5 described herein, are encompassed by the present invention. Useful fragments of the LERK-5 nucleic acids include antisense or sense oligonucleotides comprising a single strand nucleic acid sequence (either AR N or DNA) capable of binding to mRNA target sequences of LERK-5 ( sense) or DNA of LER K-5 (antisense). Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the cDNA coding region of LERK-5. Said fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on a cDNA sequence encoding a given protein, is described in, for example, Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol and others. (Biotechniques 6: 958, 1988). Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block the transcription or translation of the target sequence by any of several means, including improved degradation of duplexes, premature termination of the transcription or translation, or by other means. The antisense oligonucleotides of this form can be used to block the expression of LERK-5 proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester base structures (or other sugar linkages, such as those described in WO91 / 06629), and wherein said sugar linkages are resistant to endogenous nucleases. Said oligonucleotides with sugar-resistant bonds are stable in vivo (ie, capable of resisting enzymatic degradation), but retain the specificity of the sequence to allow binding to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides that are covalently linked to organic portions, such as those described in WO90 / 10448, and other portions that increase the affinity of the oligonucleotide for a target nucleic acid sequence., such as poly- (L-lysine). In addition, intercalators, such as ellipticine, and alkylating agents or metal complexes, can be attached to sense or antisense oligonucleotides to modify the specificity of the one ion of the antisense or sense oligonucleotide for the nucleotide target sequence. . Antisense or sense oligonucleotides can be introduced into a cell containing the target nucleic acid sequence by any method of gene transfer, including, for example, CaPO4-mediated DNA transfection, electroporation, or 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 inserting the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector which contains 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, US90 / 02656). Sense or antisense oligonucleotides can also be introduced into a cell containing the nucleotide target sequence, by the formation of a conjugate with a ligand binding molecule, as described in WO91 / 04753. Suitable ligand binding molecules, include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands to bind to cell surface receptors. Preferably, the 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 to block the entry of sense or antisense oligonucleotide, or its conjugated version , to the cell. Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing the target nucleic acid sequence by the formation of an oligonucleotide-lipid complex, as described in r-WO90 / 10448. The sense or antisense-lipid oligonucleotide complex is preferably disassociated within the cell by an endogenous lipase. Following are examples to illustrate the particular embodiments, and not to limit the scope of the invention.

EXAMPLE 1 Isolation of Human LERK-5 cDNA The cDNA encoding human LERK-5 was isolated as follows. The procedure began with the preparation of a probe to be used to identify suitable collections of cDNA for use in the cloning attempt, and to select those collections. 15 The first cDNA strand was synthesized on RNA isolated from / a variety of cell types. Then, polymerase chain reactions (PCR) were conducted by conventional techniques, using cDNAs as templates. The 5 'and 3' primers used in the PCR were oligonucleotides that define the terms of a DNA fragment of 337bp, desigd as sec label. ch 13 (GenBank® Accession No. L13819). This DNA fragment was chosen in view of its degree of homology with a portion of a Elk ligand DNA, as discussed in more detail above. A DNA band of the expected size (337bp) was amplified by PCR in a reaction where the cDNA template was derived from RNA isolated from a human T cell leukemia cell line, desigd as CCRF-HSB-2 (ATCC CCL 120.1). This single-chain 337bp DNA fragment was isolated and labeled with 32P by standard techniques to be used as a probe. Northern stains containing mRNA from a variety of tissues were probed with the 32 P-labeled DNA fragment. Hybridization mRNA (approximately 5kb) was detected in tissues that included human fetus brain and lung. In addition, the DNA bands of the expected size (337bp) were successfully amplified by PCR using, as templates, lung cDNA collections from either adult or human fetus. In this way, cDNA collections, derived from human fetus brain fibroblasts and from human lung fibroblasts, were selected for screening with the 32P labeled probe, in an effort to isolate a full length clone. The cDNA collection of human fetus brain in phage? vector? gt 10, was purchased from Clontech Laboratories, Inc., Palo Alto, California. The cDNA is inserted into the EcoRI site of the vector. The second cDNA library was derived from the human adult SV40-transformed lung fibroblast cell line, WI-26 VA4. This collection, in the phage? vector? gt 10 was constructed as described in Example 2 of the US patent. 5,264,416, which is incorporated herein by reference. Screening with the 337bp probe was conducted by conventional procedures, and hybridization clones were identified in both libraries. The nucleotide sequence of the cDNA insert of a single clone isolated from the human fetus brain collection, desigd as clone 6, was determined. The DNA sequence of the coding region of the clone? 6 cDNA, and the amino acid sequence encoded therein, are presented in SEQ ID NO: 1 and SEQ ID NO: 2. One clone was isolated from the WI26 VA4 collection composed of truncated DNA at the 5 'end, but only 5bp of initiation codon reduction of sequence 10 of SEQ ID NO: 1. The encoded protein, desigd as LERK-5, comprises an N-terminal signal peptide (amino acids 25 to 1 of SEQ ID NO: 2), an extracellular domain (amino acids 1 to 199), a transmembrane region (amino acids 200 to 225). ), and a cytoplasmic domain (amino acids 226 to 308). LERK-5 joins the two receivers of J- cell surface known as elk and hek, as demonstrated in Example 4. Nucleotides 310 to 646 of SEQ ID NO: 1 correspond to the 337bp sequence tag described above, (GenBank®, Accession No. L13819), with a pairing. He nucleotide 568 of the LERK-5 DNA of SEC I D NO: 1 is an A while a G arises in this position in the sequence tag. Possible explaons include allelic variation or cloning artifacts. If A were changed to G at position 568 of SEQ ID NO: 1, then the amino acid at position 165 could be Asp instead of Asn.

A cell lysate containing clone? 6 DNA (the cDNA of LERK-5 in? Gt 10) was deposited in American Type Culture Collection, Rockville, MD, U .S. A., on June 16, 1994, and was assigned accession number ATCC 75815. The deposit was made under the terms of the Budapest Treaty.

EXAMPLE 2 Preparation of Fusion Soluble Protein elk / Fc This example describes the construction of an expression vector encoding an elk / Fc fusion protein. This fusion protein was used in the binding analysis of Example 4, to determine if LERK-5 is capable of binding elk. A DNA and amino acid sequence encoded for rat elk cDNA was presented by Lhotak et al. (Mol.Cell. Biol.1 1: 2496, 1991), incorporated herein by reference. The rat elk protein has an extracellular domain of 538 amino acids, a transmembrane domain of 25 amino acids, and a cytoplasmic domain of 419 amino acids. A fragment of rat elk cDNA was fused to the 5 'end of cDNA encoding the Fe portion of a human IgG 1 antibody. The rat elk cDNA was obtained from T. Pawson (Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto). A restriction endonuclease cleavage site Asp718 was introduced towards the 5 'end of the elk coding region. A fragment of Asp718-Bg 1 μl of rat elk cDNA (the entire extracellular domain, the transmembrane region, and a small portion of the cytoplasmic domain) was isolated. A DNA encoding an individual polypeptide chain, comprising the Fe region of a human IgG 1 antibody, was cloned into the Spel site of the pBLU ESCRI PT SK® vector, which is commercially available from Stratagene Cloning Systems, La Jolla, California. This plasmid vector is replicable in E. Coli and contains a poly-linker segment that includes 21 unique restriction sites. The nucleotide sequence of the cloned DNA, together with the amino acid sequence of the Fe polypeptide encoded thereby, are described in the PCT application, WO93 / 10151, incorporated herein by reference. A single BglW site has been introduced, and encompasses the codons for amino acids three and four of the Fe polypeptide. The encoded Fe polypeptide extends from the N-terminal articulation region to the native C-terminus, ie, it is a Fe region. of essentially full-length antibody. The elk cDNA fragment, Asp718-BglII, was cloned into the pBLU ESCRI PT SK® vector containing the Fe cDNA, so that the elk cDNA is placed towards the 5 'end of the Fe cDNA. The strand DNA Individual, derived from the resulting fusion of the gene, was mutagenized by the method described in Kunkel (Proc. Nati, Acad. Sci. U. SA 82: 488, 1985) and Kunkel et al. (Methods in Enzymol., 154: 367, 1987). ) in order to perfectly fuse the entire extracellular domain of elk to the Fe sequence. The mutagenized DNA was sequenced to confirm that the appropriate nucleotides were removed (i.e., that the transmembrane region and the partial cytoplasmic domain DNA were deleted). ) and that the elk and Fe sequences are in the same reading frame. The elk / Fc fusion protein is preferably synthesized in mammalian host cells, such as CV1-EBNA or COS-7 cells. Fusion of the elk / Fc gene was cut and inserted into a mammalian expression vector designated HAV-EO (Dower et al J. Immunol., 142: 4314, 1989). The mammalian host cells were transfected with the resulting recombinant expression vector, and cultured to allow transient expression of the fusion protein, which was secreted into the culture medium via the elk signal peptide. The elk / Fc fusion protein was purified by affinity chromatography, using a Sepharose protein A column.

EXAMPLE 3 Preparation of Soluble Fusion Protein hek / Fc This example describes the construction of an expression vector encoding a hek / Fc fusion protein. This fusion protein was used in the binding analysis of Example 4, to determine if LERK-5 is capable of joining hek. A DNA and amino acid sequence encoded for human hek cDNA was presented by Wicks et al. (Proc. Nati, Acad. Sci. U.S.A., 89: 161 1, 1992), incorporated herein by reference. This hek protein comprises (from the term N- to C-) an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Two DNA fragments, one encoding an N-terminal fragment of the extracellular domain of hek and the other encoding a C-terminal fragment of the extracellular domain of hek, were isolated by polymerase chain reactions (PCR) conducted under normal conditions, using primers of oligonucleotide based on the nucleotide sequence of hek published by Wicks et al., supra. The template for the PCRs was cDNA prepared from mRNA isolated from a human T cell leukemic cell line designated as CCRF-HSB-2 (ATCC CCL-120.1). The PCR products containing the 5 'end of the hek DNA were digested with Spel and Hindl ll to isolate a DNA fragment extending from the 5' end of the mature human hek sequence (i.e. encodes the signal sequence) to a Hindl ll site, found in the hek gene. PCR products containing the 3 'end of the hek extracellular domain DNA were digested with Hindl lly Clal to isolate a fragment extending from the inner site of Hindl ll to a Clal site just downstream of the 3' end of the sequence which encodes the extracellular domain of hek. The Clal site is a multiple cloning site (month) introduced just towards the 3 'end of the extracellular domain. A DNA encoding a human Fe chain of a human IgG 1 antibody was encoded. This Fe mutein DNA and the polypeptide encoded by it, are described in the patent application of E. U.A. series No. 08 / 097,827, entitled "Novel Cytokine Which is a Ligand for OX40" ("Novel Cytokine which is a Ligand for OX40"), filed on July 23, 1993, which is incorporated herein by reference. The mutein DNA was derived from a DNA encoding the native Fe polypeptide by site-directed mutagenesis, conducted essentially as described by Deng and Nickoloff, Anal, Biochem, 200: 81 (1992). The amino acid sequence of the Fe mutein polypeptide is identical to that of the native Fe polypeptide described in PCT application WO93 / 10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu. to Glu, and amino acid 22 has been changed from Gly to Ala. This Fe of mutein exhibits a reduced affinity for immunoglobulin receptors. A recombinant vector, containing the mutein DNA of Fe, was separated with Clal and Notl, which separate the vector in a poly-linker region immediately towards the 5 'end and towards the 3' end, respectively, of the mutein DNA insert. Fe. The desired fragment encoding Fe mutein was isolated. Mutein Fe polypeptide extends from the N-terminal articulation region to the native C-terminus, ie, it is an antibody Fe region of essentially complete length . Fragments of the Fe, v regions may also be used. gr. , those that are truncated at the C-terminal end. The fragments preferably contain multiple residues of cysteine (at least the cysteine residues in the joint reaction) to allow cross-disulfide bonds to form between the portions of the Fe polypeptide and two separate hek / Fc fusion proteins, creating dimers. A mammalian expression vector designated SMAG4 was separated with Spel and Notl. The SMAG4 vector comprises a sequence encoding the murine interleukin-7 signal peptide (described in the U.S. Patent 4,965, 195) inserted into the mammalian high expression vector., pDC202 (described by Sims et al., Science 242: 585, 1988, and in PCT application WO89 / 03884), which is also capable of replication in E. coli. Spel separates the vector immediately towards the 3 'end of the sequence encoding the signal peptide I L-7. Notl separates approximately 155bp towards the 3 'end of the Spel site at a multiple cloning site of the vector. The Spel / Notl fragment, which contains the vector sequences and the DNA encoding signal peptide I L-7, was isolated. A ligation of four forms was conducted to insert the two DNA fragments encoding hek and the DNA fragment encoding the Fe mutein, described above, into the SMAG4 expression vector separated by Spel / Notl. E. Coli cells were transfected with the ligation mixture and the desired recombinant vector was isolated therefrom. The isolated vector encodes a fusion protein comprising (from the N- to C- terminus) the murine IL-7 signal peptide, the extracellular domain of hek, four amino acids encoded by the introduced month, and the Fe mutein. , the expression vector was co-transfected with the plasmid pSV3.NEO into the CV1 / EBNA cells. The cell line of CV1 / EBNA (ATCC CRL 10478) was derived from a monkey kidney cell line, as described by McMahan et al. (EMBO J. 10: 2821, 1991). The vector pSV3.NEO expresses the SV40 T antigen, which is not produced by the host cells. The vector pSV3.NEO is similar to pSV3 (Mulligan and Berg, Proc. Nati, Acad. Sci. U.S.A. 78: 2072, 1981), but also contains a neomycin resistance gene. Transformed cells were cultured to allow transient expression of the fusion protein, which is secreted into the culture medium via the murine IL-7 signal peptide. The fusion protein was purified on a protein A sepharose column, eluted, and used to screen cells for hek / Fc protein binding ability, as described in Examples 2 and 3.

EXAMPLE 4 Linkage Study The ability of LERK-5 to bind to receptors, known as elk and hek, was analyzed in the following analysis: The procedure began with the preparation of expressing cells LERK-5 on the surface of the cell. The LERK-5 DNA was amplified by PCR, using the DNA of clone? 6, described in Example 1, as a template. The primers used in the PCR defined the terms of the coding region of the LERK-5 DNA, and were also added to an Xhol restriction site at the 5 'end and a NotI site in the end 3 'of the amplified DNA. The 5 'primer was further added to a consensus Kozak sequence towards the 5' end of the initiation codon. The reaction products were digested with Xhol and Notl and inserted into a separate expression vector with Sali ( which is compatible with Xhol) and Notl. The expression vector was pDC410, which is a mammalian expression vector that also replicates in E. Coli. pDC410 is similar to pDC406 (McMahan et al., EMBO J. 10: 2821, 1991) the multiple cloning site of pDC410 (month) differs from that of pDC406, in that it contains sites of additional restriction and three retention codons (one in each s? - m reading arch). A T7 polymerase promoter towards the 3 'end of the month facilitates the sequence formation of the inserted DNA towards the month. In addition, the EBV origin of replication is replaced by the DNA encoding the SV40 large antigen T (derived from a SV40 promoter) in pDC410. CV1-EBNA-1 cells were transfected in 10cm2 dishes with the recombinant expression vector containing LERK-5 DNA. The cell line CV-1 / EBNA-1 (ATCC CRL 10478) constitutively expresses EBV nuclear antigen-1 immediately old CMV enhancer / promoter. CV-1-EBNA-1 was derived from CV-1 of the kidney cell line of the African green monkey (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10: 2821, 1991. Transfected cells were cultured for 24 hours, and the cells in each plate were divided into a 24-well plate.After culturing for an additional 48 hours, a binding analysis was conducted by the following procedure: Transfected cells (approximately 4 x 10 4 cells / cavity) were washed with BM-NFDM, which is a binding medium (RPMI 1640 containing 25 mg / ml of bovine serum albumin, 2 mg / ml of sodium azide, 20 mM of Hepes at a pH of 2.7). ), to which 50 mg / ml of fat-free dry milk was added, then the cells were incubated for 1 hour at room temperature with various concentrations of the elk / Fc fusion protein, prepared in Example 2, or the fusion protein of hek / Fc, prepared in Example 3. Then, the cells were washed and incubated with a constant saturation concentration of a 12Sl-mouse anti-human IgG in a binding medium, with moderate agitation for 1 hour at room temperature. After extensive washing, the cells were released via trypsinization. The mouse anti-human IgG employed above is directed against the Fe region of human IgG and was obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody was radioiodinated using the normal chloramine-T method. The antibody will bind to the Fe portion of any of the elk / Fc or hek / Fc fusion proteins, which has bound to the cells. In all analyzes, a non-specific binding of the 125I-antibody was analyzed in the absence of elk / Fc (or hek / Fc), as well as in the presence of elk / Fc (or hek / Fc) and a 200-fold molar excess of the anti-mouse IgG non-labeled antibody. The 125I-antibody bound to the cell was quantified in a Packard Autogamma counter. Affinity calculations were generated (Scatchard, Ann. N. Y. Acad. Sci. 51: 660, 1949), using a Delta plot. In the analysis to bind elk / Fc, cells expressing recombinant LERK-5 exhibited a single affinity binding class, with approximately 103,317 binding sites per cell. The affinity constant (Ka) was 1.05 x 10s M * P When the results of the four binding analyzes were averaged, the Ka of the elk / Fc binding was 1.2 + 0.3 x 109 M_P. found that the cells, which express LERK-recombinant, bind hek / Fc. The Ka of the union of hek / Fc was 4.3 + _3.3 x 107 M "1 (average of the four experiments).

EXAMPLE 5 Northern Staining Analysis To investigate the expression of LERK-5 in various tissues, Northern stains were tested, which contained mRNA from a variety of human tissues (both fetal and adult) with a riboprobe of LERK-5. The riboprobe was derived from the 337 bp DNA fragment, isolated in Example 1, as follows. Oligonucleotides, which define the terms of the 337bp DNA fragment, were used as primers in a polymerase chain reaction (PCR). The 3 'primer also included the T3 RNA polymerase promoter. PCR was carried out by conventional techniques, using the 337bp DNA as the template. The amplified DNA thus obtained contained the T3 RNA polymerase promoter, at the 3 'end. A riboprobe was prepared by normal techniques, using T3 RNA polymerase and the amplified DNA, as the template. The stains were hybridized with the riboprobe at 63 ° C, then washed, first with 1 XSSC / 0.1% SDS for 1 hour at 68 ° C, then with 0.1 XSSC / 0.1% SDS for 30 minutes at 68 ° C. The stain was exposed to an X-ray film at -70 ° for 11 days, sandwiched between two intensification screens. In staining containing RNA derived from fetal tissues, a larger band of mRNA, approximately 5.0 kb, was visualized in the heart, brain, lung, and kidney, but not in the liver. In stains containing RNA from adult tissues, a 5 kb band was detected in the lung and kidney, although expression levels emerged lower than in fetal tissues. No band of mRNA hybridization was detected, or was very weak, in the adult heart, brain, placenta, liver, skeletal muscle, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and lymphocytes. peripherally blood.

EXAMPLE 6 Monoclonal Antibodies for LERK-5 This example illustrates the preparation of monoclonal antibodies for LERK-5. LERK-5 is expressed in mammalian host cells such as COS-7 cells, or CV-1 / EBNA-1, and purified using elk / Fc affinity chromatography. Purified LERK-5 (or a fragment thereof, such as the extracellular domain) can be used to generate monoclonal antibodies against LERK-5, using conventional techniques, for example, those techniques described in the U.A. 4, 41 1, 993. Briefly, mice were immunized with LERK-5, as an immunogen emulsified in Freund's complete adjuvant, and injected in amounts ranging from 10-100 μg, subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals were reinforced with additional LERK-5 emulsified in Freund's incomplete adjuvant. The mice were periodically reinforced afterwards, in a weekly to biweekly immunization schedule. Serum samples were periodically taken by retro-orbital bleeding or by a cut at the tip of the tail, to be tested, by spot staining analysis or ELISA (Enzyme-Linked Immunosorbent Assay), for LER K-5 antibodies. After detection of an appropriate antibody concentration, the positive animals were given one last injection of LERK-5 in saline. Three to four days later, the animals were sacrificed, the spleen cells harvested, and the spleen cells were fused to a murine myeloma cell line, e.g. , NS 1 or preferably P3x63Ag8.653 (ATCC CRL 1580). The fusions generate hybridoma cells, which were placed in multiple microtiter plates in a selective medium of HAT (hypoxanthine, aminopterin and thymidine), to inhibit the proliferation of unfused cells, myeloma hybrids, and spleen cell hybrids. Hybridoma cells were screened by ELISA for reactivity against purified LERK-5, by adaptations of the techniques described by Engvall et al., Immunochem. 8: 871, 1981 and in the patent of E. U.A. 4,703, 004. A preferred screening technique is the antibody capture technique described by Beckmann et al. (J. Immunol., 144: 4212, 1990). The positive hybridoma cells can be injected intraperitoneally into syngeneic BALB / c mice, to produce spots containing high concentrations of anti-LERK-5 monoclonal antibodies. Alternatively, hydridoma cells can be grown in vitro in flasks or spinning bottles by various techniques. Monoclonal antibodies produced in mouse asitoses can be purified by precipitation of ammonium sulfate, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on binding of the antibody to protein A or protein G, as well as affinity chromatography based on binding to LERK-5 can also be used.

EXAMPLE 7 Receptor Phosphorylation In the following experiment, LERK-5 demonstrated the ability to induce elk phosphorylation. A soluble protein of LERK-5 / Fc was prepared by fusing DNA encoding amino acids, from 25 to 198, of LERK-5 of SEQ ID NO: 2 to the DNA encoding the human IgG 1 polypeptide Fe, described above, in the vector pDC303. The mammalian expression vector pDC303, also known as SF CAV, is described in the PCT application, WO93 / 19777. The soluble protein of LERK-5 / Fc was expressed and purified by means of procedures analogous to those described by Fanslow et al. (J. Immunol., 149: 655, 1992). Rat elk cDNA was inserted into the mammalian expression vector, pDC303, and transfected into CV1 / EBNA cells. The cells were separated from a 10 cm plate to a 6-well plate after 24 hours. After two additional days, the cells were stripped of methionine and cysteine, and then radiolabeled for 3 hours with a mixture of 1: 1 of [3SS] methionine and [3SS] cysteine (Amersham) at a total concentration of 100 μCi ml "P Stimulations were performed by adding soluble LERK-5Fc or soluble elk / L / Fc (described in WO94 / 1384) at 1 μg mi" 1 for 10 minutes at 37 ° C. The cells were then rapidly washed 2X with PBS + 1 mM orthovanadate, cold, and lysed in lysing pH regulator (25 mM tris HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM fluoride of Na, 1% NP-40, 1 mM DTT, 1 mM orthovanadate, plus protease inhibitors). Cell lysates were immunoprecipitated with a rabbit anti-elk antibody using protein G sepharose (Sepharose). The anti-elk antibody was generated by immunizing rabbits with a human elk-Fc fusion protein. After washing 3X with pH regulator of RI PA (50 mM of H EPES, pH 8.0, 150 mM of NaCl, 0.5% of sodium deoxycholate, 0.5% of nonidet P-40 and 0.1% SDS), and 1 X with PBS, the samples were placed in a reducing pH buffer solution and analyzed by SDS-PAGE. Then, the proteins were transferred to nitrocellulose and proteins marked with [3SS] were detected on a Phophorlmager (Molecular Dynamics). The membrane was then immunostained with anti-phosphotyrosine 4G10 (Upstate Biotech Inc., Lake Placid, NY) followed by biotin-goat anti-mouse (Kirkegaard and Perry Research Laboratories, Inc., Gaithersburg, MD), streptavidin-peroxidase ( Kirkegaard and Perry), and then revealed using SL development reagents (Amersham). Western analysis of elk immunoprecipitates with anti-phosphotyrosine antibody showed that LERK-5 / Fc stimulated elk phosphorylation, as well as for elk-L / Fc. No ligand-dependent phosphorylated proteins were detected in CV1 / EBNA cells treated with only the medium or transfected with a control plasmid. f. > LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTS: Immunex Corporation 5 (ii) TITLE OF THE NTION: Novel Cytokine Designated as LERK-5 (iii) SEQUENCE NUMBER: 3 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Immunex Corporation 10 (B) STREET: 51 University Street (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: USA (F) AREA: 98101 15 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEANS: Flexible disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Apple 7.1 (D) SOFTWARE: Microsoft Word, Version 5.1a 20 (vi) REAL DATA OF THE APPLICATION: (A) APPLICATION NUMBER: - will be assigned - ( B) DATE OF SUBMISSION: July 6, 1995 (C) CLASSIFICATION: (vii) DATA OF THE PREVIOUS APPLICATION: 25 (A) NUMBER OF APPLICATION: US 08 / 271,948 (B) DATE OF SUBMISSION: July 8, 1994 (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Anderson, Kathryn A. (B) REGISTRATION NUMBER: 32,172 (C) REFERENCE NUMBER / DOC .: 2823-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (206) 587-0430 (B) TELEFAX: (206) 233-0644 (C) TELEX: 756822 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1002 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (¡) i) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B) CLON: huLERK-5 (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 1..1002 (ix) ASPECT: (A) NAME / KEY: sig_peptide (B) LOCATION: 1 ..75 (ix) ASPECT: (A) NAME / KEY: mat_peptide (B) LOCATION: 76 ..999 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG GCT GTG AGA AGG GAC TCC GTG TGG AAG TAC TGC TGG GGT GTT TTG 48 Met Wing Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu -25 -20 -15 -10 ATG GTT TTA TGC AGA ACT GCG ATT TCC AAA TCG ATA GTT TTA GAG CCT 96 Met Val Leu Cys Arg Thr Wing He Ser Lys Ser He Val Leu Glu Pro -5 1 5 ATC TAT TGG AAT TCC TCG AAC TCC AAA TTJ CTA CCT GGA CAGA GGA CTG 144 He Tyr Trp Asn Ser Being Asn Being Lys Phe Leu Pro Gly Gln Gly Leu 10 15 20 GTA CTA CAC CAG CAG ATA GGA AAA TTG GAT ATT ATT TGC CCC AAA '192 Val Leu Tyr Pro Gln He Gly Asp Lys Leu Asp He He Cys Pro Lys 25 30 35 GTG GAC TCT AAA ACT GTT GGC CAG TAT GAA TAT TAT AAA GTT TAT ATG 240 Val Asp Ser Lys Thr "'al Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met 40 45 50 55 GTT GAT AAA GAC CA GCA GAC AGA TGC ACT ATT AAG AAG GAA AAT ACC 288 Val Asp Lys Asp Gln Wing Asp Arg Cys Thr He Lys Lys Glu Asn Thr 60 65 70 CCT CTC CTC AAC TGT GCC AAA CCA GAC CAA GAT ATC AAA TTC ACC ATC 336 Pro Leu Leu Asn Cys Wing Lys Pro Asp Gln Asp He Lys Phe Thr He 75 80 85 AAG TTT CAA GAA TTC AGC CCT AAC CTC TGG GGT CTA GAA TTT CAG AAG 384 Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln Lys 90 95 100 AAC AAA GAT TAT TAC ATT ATA TCT ACA TCA AAT GGG TCT TTG GAG GGC 432 Asr. Lys Asp Tyr Tyr He He Ser Thr Ser Asn Gly Ser Leu Glu Gly 105 110 115 CTG GAT AAC CAG GAG GGA GGG GTG TGC CAG ACA AGA GCC ATG AAG ATC 480 Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys He 120 125 130 • 135 CTC ATG AAA GTT GGA CAA GAT GCA AGT TCT GCT GGA TCA ACC AGG AAT 528 Leu Met Lys Val Gly 31n Asp Ala Ser Ser Ala Gly Ser Thr Arg Asr. 140 145 150 AAA GAT CCA ACA AGA '; GT CCA GAA CTA GAA GCT GGT ACA AAT GGA AGA 576 Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Wing Gly Thr Asn Gly Arg 155 160 165 AGT TCG ACA ACA AGT CCC TTT GTA AAA CCA AAT CCA GGT TCT AGC ACA 624 Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Be Ser Thr 170 175 180 GAC GGC AAC AGC GCC GGA CAT TCG GGG AAC AAC ATC CTC GGT TCC GAA 672 Asp Gly Asn Be Wing Gly His Ser Gly Asn Asn He Leu Gly Ser Glu 185 190 195 GTG GCC TTA TTT GCA GGG ATT GCT TCA GGA TGC ATC ATC TTC ATC GTC 720 Val Ala Leu Phe Ala Gly He Ala Be Gly Cys He He Phe He Val 200 205 210 215 ATC ATC ATC ACG CTG GTG GTC CTC TTG CTG AAG TAC CGG AGG AGA CAC 768 He He He Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg Arg His 220 225 230 AGG AAG CAC TCG CCG CAG CAC ACG ACC ACG CTG TCG CTC AGC ACA CTG 816 Arg Lys His Ser Pro .-- In His Thr Thr Thr Leu Ser Leu Ser Thr Leu 235 240 245 GCC ACA CCC AAG CGC; .3C GGC AAC AAC AAC GGC TCA GAG CCC AGT GAC 864 Wing Thr Pro Lys Arg Gly Asn Asn Asn Gly Ser Glu Pro Ser Ase 250 255 260 ATT ATC ATC CCG CTA AGG ACT GCG GAC AGC GTC TTC TGC CCT CAC TAC 912 He He He Pro Leu Arg Thr Wing Asp Ser Val Phe Cys Pro His Tyr 265 270 275 GAG AAG GTC AGC GGG GAC TAC GGG CAC CCG GTG TAC ATC GTC CAG GAG 960 Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr He Val Gln Glu 28C 285 290 295 ATG CCC CCG CAG AGC CCG GCG AAC ATT TAC TAC AAG GTC TGA 1002 Met Pro Pro Gln Ser Pro Wing Asn He Tyr Tyr Lys Val 300 305 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 333 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: protein (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Wing Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu -25 -20 -15 -10 Met Val Leu Cys Arg Thr Wing Me Ser Lys Ser He Val Leu Glu Pro -5 1 5 He Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 10 15 20 Val Leu Tyr Pro Gln He Gly Asp Lys Leu Asp He He Cys Pro Lys 25 30 35 Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met 40 45 50 55 Val Asp Lys Asp Gln Wing Asp Arg Cys Thr He Lys Lys Glu Asn Thr 60 65 70 Pro Leu Leu Asn Cys Wing Lys Pro Asp Gln Asp He Lys Phe Thr He 75 80 85 Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu .Phe Gln Lys 90 95 100 Asn Lys Asp Tyr Tyr He He Ser Thr Ser Asn Gly Ser Leu Glu Gly 105 110 115 Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys He 120 125 130 135 Leu Met Lys Val Gly Gln Asp Wing Being Ser Wing Gly Ser Thr Arg Asn 140 145 150 Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Wing Gly Thr Asn Gly Arg 155 160 165 Be Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr 170 175 180 Asp Gly Asn Ser Wing Gly His Ser Gly Asn Asn He Leu Gly Ser Glu 185 190 195 Val Ala Leu Phe Wing Gly He Wing Ser Gly Cys He He Phe He Val 200 205 210 215 He He He Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg Arg His 220 225 230 Arg Lys His Ser Pro Gln His Thr Thr Thu Leu Ser Leu Ser Thr Leu 235 240 245 Wing Thr Pro Lys Arg Ser Gly Asn Asn Asn Gly Ser Glu Pro Ser Asp 250 255 260 He He He Pro Pro Leu Arg Thr Wing Asp Ser Val Phe Cys Pro His Tyr 265 270 275 Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr He Val Gln Glu 280 285 290 295 Met Pro Pro Gln Ser Pro Wing Asn He Tyr Tyr Lys Val 300 305 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRUCTURE CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B) CLON: peptide FLAG (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims (10)

1. - An isolated DNA encoding a protein of LERK-5 able to bind elk and hek, wherein said LERK-5 comprises an amino acid sequence selected from the group consisting of amino acids 25 to 308 of SEQ ID NO: 2, and 1- 308 of SEQ ID NO:

2. 2. A DNA according to claim 1, wherein said DNA comprises a nucleotide sequence selected from the group consisting of nucleotides 1 to 1002 of SEQ ID NO: 1 and nucleotides 76 to 1002 of SEQ ID NO: 1.

3. An isolated DNA encoding a soluble human LERK-5 protein capable of binding elk and hek, wherein said LERK-5 comprises an amino acid sequence selected from the group consisting of amino acids 25 to 199 of SEQ ID NO: 2, and 1-199 of SEQ ID NO: 2.

4. A DNA according to claim 3, wherein said DNA comprises a nucleotide sequence selected from the group consisting of nucleotides 1 to 672 of SEQ ID NO: 1 and nucleotides 76 to 672 of SEQ ID NO: 1.

5. An isolated DNA encoding a soluble LERK-5 polypeptide capable of binding elk and hek, wherein said soluble LERK-5 polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of residues 1 -199 of SEQ ID NO: 2.

6. An isolated DNA that encodes a LERK-5 polypeptide capable of binding elk and hek, wherein said LERK-5 polypeptide comprises an extracellular domain, a transmembrane region, and a cytoplasmic domain, and wherein said polypeptide LERK-5 comprises an amino acid sequence that is at least 90% identical to the residue sequence 1 -308 of SEQ ID NO: 2.

7. An expression vector comprising a DNA according to claim 1.

8. An expression vector comprising a DNA according to claim 3.

9. An expression vector comprising a DNA according to claim 5.

10. An expression vector comprising a DNA according to claim 6. 1 1 .- A method for preparing a LERK-5 polypeptide, comprising culturing a host cell transformed with a vector according to claim 7 under LERK-5 expression promoting conditions, and recovering the polypeptide from LERK-5 of the crop. 12. A method for preparing an LER K-5 polypeptide, comprising culturing a host cell transformed with a vector according to claim 8 under LERK-5 expression promoting conditions, and recovering the polypeptide from LERK-5 of the crop. 13. A method for preparing a LERK-5 polypeptide, comprising culturing a host cell transformed with a vector according to claim 9 under LERK-5 expression promoting conditions, and recovering the polypeptide from LERK-5 of the crop. 14. A method for preparing a LERK-5 polypeptide, comprising culturing a host cell transformed with a vector according to claim 10 under LERK-5 expression promoting conditions, and recovering the polypeptide from LERK-5 of the crop. 15. A mature, purified, human LERK-5 protein capable of an ir elk and hek, wherein said human LERK-5 protein is characterized by the N-terminal amino acid sequence Lys, Ser, Me, Val, Leu, Glu, Pro, Lie, Tyr, Trp, Asn, Ser. 16. The purified LERK-5 protein according to claim 15, wherein said LERK-5 comprises an amino acid sequence selected from the group consisting of amino acids 1 - . 1 -308 of SEC I D NO: 2, and 1 -199 of SEC I D NO: 2. 17. A purified, purified LERK-5 polypeptide, wherein said polypeptide is encoded by a DNA according to claim 5. 18. A purified, purified LERK-5 polypeptide, wherein said polypeptide is encoded by a DNA according to claim 6. 19. An antibody that is immunoreactive with a human LERK-5 polypeptide according to claim 16. 20. An antibody according to claim 19, wherein said antibody is a monoclonal antibody. 21. A fusion protein comprising a soluble human LERK-5 polypeptide according to claim 17, and a Fe polypeptide. 22. A dimer comprising two fusion proteins according to claim 21, wherein said fusion proteins are linked by disulfide bonds.