KR19990087793A - Cytochin labeled as Luke-8 - Google Patents

Abstract

The present invention is directed to a novel site labeled as LUCK-8. Luck-8 binds to cell surface receptors known as hecks and elk, which are members of the F / Elk family of tyrosine kinase receptors.

The purified Lurk-8 protein is provided with an isolated DNA encoding Lurk-8 in the body, an expression vector comprising Lurk-8 DNA and a host cell transformed with the expression vector. Production methods of Lurk-8 include culturing modified host cells under conditions that promote expression of Lurk-8 polypeptides and recovering Lurk-8. The invention also encompasses antibodies directed against Luck-8.

Description

Cytochin labeled as Luke-8

Proteins known as tyrosine kinase receptors have intrinsic kinase activity that is active in ligand binding. This type of protein is characterized by conserved structural motifs in the catalytic domain (Hanks et al., Science, 242: 42, 1988), and is characterized by structural features of the N- Family).

The receptor for the receptor named after the first member isolated (Hirai et al. Science 238: 1717, 1987) is the largest subspecies of tyrosine kinase receptors. Among these members are Chicken Cheek 4 (Sajjadi et al. New Biol. 3: 769, 1991) and Chek 5 (Pasquale, E. B., Cell Regulation 2: 523, 1991); (Zenk et al., J. Neurosci. Res., 37: 129, 1994), nuk (Henkemeyer et al Oncogene 9: 1001, 1994) and sek (Gilardi-Hebenstreit et al., Oncogene 7: 2499, 1992); Eck (Chan et al., Oncogene 6 (1999)), the rat elk (Letwin et al., Oncogene 3: 621, 1998; Lhotak et al., Mol. : 1057, 1991), ehk-1 and eeq-2 (Maisonpierre et al., Oncogene 8: 3277, 1993); And human hek (Boyd et al., J. Biol. Chem., 267: 3262, 1992; Wicks et al., PNAS USA, 89: 1611, 1992) 8: 2857,1993), Eck (Lindberg et al., Mol. Cell Biol. 10: 6316,1990) and erk (Chan et al., Supra).

These subfamily proteins are involved not only in their cytoplasmic regions but also in their extracellular regions, with 41-68% being identical. Interestingly, the tissue distribution of these different receptors varies. Since many FEF-associated tyrosine kinase receptors are expressed primarily in the brain, it has been thought that these receptors and their ligands may be associated with the growth, differentiation and survival of neurons.

Ligands that have been identified as tyrosine kinase receptors are a group of proteins that affect the growth, differentiation and survival of cells expressing receptors. It has been found that certain ligands bind to one or more receptors of the ff. Examples of ligands of Hek and elk are described below.

The identification of additional ligands for Hek and elk that may be present will prove to be useful in investigating the properties of cellular processing modulated by signals through these receptors. If it is desired to increase or suppress specific physiological signals mediated through these receptors, it is beneficial to identify each of the proteins that can serve to transduce such signals. Furthermore, interleukin-1 receptor antagonist proteins (Eisenberg et al., Nature 343: 341, 1990; Hannum et al., Nature 343: 336, 1990; and Carter et al., Nature 344: 633, 1990) Are known to be capable of binding to receptors without the initiation of signal transduction. The identification of additional proteins binding to a HEK or an ELK is also desirable to determine which of those proteins function as antagonists.

The present invention provides novel cytokines designated Lurk-8. This cytokine binds to the elk and the known tyrosine kinase receptor.

The present invention includes a DNA encoding Lurk-8, an expression vector comprising Lurk-8 DNA, and a host cell transformed with an expression vector. A method of producing a Lurk-8 polypeptide comprises culturing a transformed host cell under conditions that induce expression of Lurk-8, and recovering the expressed Lurk-8. Purified Lurk-8 polypeptides that are in soluble and membrane bound form are disclosed.

A Lurk-8 polypeptide or an immunogenic fragment thereof can be used as an immunogen to produce antibodies that are immunoreactive. One of the embodiments of the present invention is that the antibody is a monoclonal antibody.

The cDNA encoding human Lurk-8 was isolated as described in Example 1. The nucleotide sequence of this Lurk-8 cDNA is present in SEQ ID NO: 1 and the encoded amino acid sequence is present in SEQ ID NO: This Lurk-8 protein contains an N-terminal signal peptide (amino acid -27 to -1), an extracellular domain (amino acids 1 to 197), a trans-membrane region (amino acids 198 to 224), and a cytoplasmic domain (amino acids 225 to 313) .

The calculated molecular weight of the mature human Lurk-8 protein (SEQ ID NO: 2 amino acids 1 to 313) is about 33 kilodaltons and the isoelectric point (pI) is 8.46. Thus, an example of the present invention relates to a method for producing an amino acid sequence comprising the amino acid sequence of Leu-Ser-Leu-Glu-Pro-Val-Tyr-Trp-Asn- Ser-Ala- Asn- (SEQ ID NO: 1-12), a purified human Lurk-8 protein with a calculated molecular weight of about 33 kilodaltons and an isoelectric point of 8.46. The calculated molecular weight is based on the molecular weight of the protein of a particular amino acid sequence except for glycosylation. Those skilled in the art will recognize that the glycosylated form of the protein will have a higher molecular weight.

Lack-8 fragments, such as fragments that retain the ability to associate with a heck or elk, are also provided. An example of such a fragment is a soluble Lurk-8 polypeptide.

The present invention provides both cell membrane binding and soluble (secreted) forms of Lurk-8. The soluble Lurk-8 polypeptide contains the receptor-binding domain of Lurk-8, but lacks the trans-membrane portion that can cause it to have a polypeptide on the cell membrane. As one example, soluble luc-8 comprises the entire extracellular domain (e. G., Amino acids 1 to 197 of human luc-8 of SEQ ID NO: 2). In other cases, the soluble polypeptide is a fragment of the Lurk-8 extracellular domain with the ability to bind to the Alk and Heck. The portion of the extracellular domain believed to be most important in receptor binding comprises amino acids 1-142 of SEQ ID NO: The rest of the extracellular domain (amino acids 143-197) constitutes the spacer moiety. Examples of soluble human luc-8 polypeptides include, but are not limited to, C-terminal truncated polypeptides that are either residues of residues 142-197 of SEQ ID NO: 2 or residues at that position . In other words, such soluble Lurk-8 polypeptides comprise amino acids 1 to y of SEQ ID NO: 2, wherein y is an integer between 142 and 197.

Soluble Lurk-8 can be identified by testing the medium (supernatant) for the presence of the desired protein by isolating complete cells expressing the Lurk-8 polypeptide from the culture medium, such as, for example, by centrifugation Soluble membrane bonds). The presence of Luck-8 in the medium indicates that the protein is secreted from the cell and thus is in soluble form of the desired protein.

The soluble form of Luck-8 has certain advantages over the membrane-bound form of the protein. Since the soluble protein is secreted from the cell, the purification of the protein from the recombinant host cell is facilitated. Moreover, soluble proteins are generally more suitable for certain applications, such as, for example, intravenous administration methods.

When initially expressed in a host cell, the soluble Lurk-8 polypeptide advantageously comprises one of the following signal peptides or amorphous leaders functional in the host cell used or a natural signal peptide. An isolated DNA sequence encoding the soluble Lurk-8 protein is included in the present invention.

The cleaved LUC-8 comprising soluble polypeptides can be prepared by one of a variety of conventional techniques. DNA sequences encoding truncated Lurk-8 can be chemically synthesized using known techniques. DNA fragments can also be produced by restriction of endonuclease digestion of the full length cloned DNA sequence and can be isolated by electrophoresis on an agarose gel. Oligonucleotides in which the 5 ' or 3 ' -end of the DNA fragment is reproduced at the desired site can be used. LINKERS containing restriction of the endonuclease cleavage site can be used to insert the desired DNA fragment into an expression vector. Well known polymerase chain reaction (PCR) methods can also be used to amplify DNA fragments encoding specific protein fragments. Primers that define the desired end of the DNA fragment are used in PCR. Alternatively, known mutagenesis techniques can be used to insert a stop codon immediately at the desired site, e.g., at the bottom of the codon of the last amino acid of the receptor binding domain.

The expressed sequence label (EST) contains the region identified by SEQ ID NO: 1 (see Example 1). The computer data bank record for this EST (accession number H10006) represents the DNA sequence 454 nucleotides in length. When the EST H10006 sequence was designated as SEQ ID NO: 1, the identified site was found between nucleotides 663 and 1118 of SEQ ID NO: The constant nucleotides in EST H10006 are not identifiable (i. E., Designated as " N " in the databank record because its identification is not known). The EST sequence inserts nucleotides not found at corresponding positions in SEQ ID NO: 1 and contains deletion and mismatch when compared to SEQ ID NO:

No reading frame is identified in the data bank file for EST, and the sequence lacks an initiation codon. Moreover, the insertion and deletion will result in movement in the leading frame compared to the reading frame of the sequence No. 1, Lurk-8 sequence. However, even if the reading frame is clarified or identified, and inserted, removed and unmodified, the translation of EST H10006 will result in one of the four cysteine residues conserved in the other LUCK proteins (described below) And likewise lacks other conserved residues. Four conserved cysteines are believed to be important in their binding properties to elk and hek.

Other proteins associated with both HEK and the elk were found, and LUCK-1 was shown via Lurk-7 (a ligand of an F-related kinase). Luck 2 and 5 are type 1 trans-membrane proteins (Luck-8), whereas Lucks 1,3,4,6 and 7 are very close to the cell membrane by the GPI chain. The percentage of amino acid sequence of these six proteins is 24 to 59%, and each protein has four conserved cysteine residues.

Holtsman et al. (Mol. Cell. Biol. 10: 5830, 1990) reported the coding of a protein called B61. The ability of B61 to associate with elk and heck was subsequently discovered, and the B61 protein was given as an optional display L-1 (Beckmann et al., EMBO J. 13: 3757, 1994). B61 has also been reported as a ligand for the above tyrosine kinase receptor known as Eck (Bartley et al., Nature 368: 558, 1994).

In addition, Luck-2, known as an elk ligand, is described in PCT application WO 94/11384. In addition, all of Luck-3 and Luck-4 known as Hek ligands are described in PCT application WO 95/06065, Luck-5 in WO 96/01839, Luck-6 in WO 96/10911, Luck-7 Are described in WO 96/17925.

The percent identity of the human luc-8 amino acid sequence of SEQ ID NO: 2 having the full length amino acid sequence of various other proteins is as follows. Here, h represents a human, m represents a mouse, and r represents a rat.

h Luck-1 25.14

h Luck-2 40.80

ruck-2 39.69

m Luck -2 40.00

h Luck-3 24.88

h Luck -4 25.41

h Luck-5 41.23

m Luck -5 42.07

m Luke -6 26.18

h Luck -7 24.88

As used herein, the term " Luck-8 " refers to the type of polypeptide that is substantially homologous to the human Lurk-8 protein described in Example 1. Preferably, the polypeptide comprises an amino acid sequence that is at least 80% identical, more preferably at least 90% identical, to the amino acid sequence of SEQ ID NO: 2, as further described below. The Lurk-8 polypeptide may bind to the receptor expressed in HEK and ELK. More specifically, as described below, certain uses of LUCK-8 result from this ability to combine with elk or heck. Human luc-8 nucleic acids and proteins are within the scope of the present invention, such as Lurk-8 nucleic acids and proteins obtained from other mammalian species including, but not limited to, rats, cows, pigs, horses or several primate species.

Due to the known homology of the genetic code encoding one or more codons for the same amino acid, the DNA sequence can vary from that shown in SEQ ID NO: 1, But still encodes the Lurk-8 protein with the amino acid sequence. Such modified DNA sequences may be generated from silent mutations (e. G., Occurring during PCR amplification) or may also be silent mutagenic products of the native sequence. Thus, the present invention contemplates the use of an isolated DNA sequence selected from a native Lurk-8 DNA sequence (e.g., a cDNA comprising the nucleotide sequence present in SEQ ID NO: 1) and a genetic code for the native Lurk-8 DNA sequence Provides DNA that is shrunk.

The Lurk-8 polypeptides provided herein contain variants of natural Lurk-8 polypeptides that retain the biological activity of native Lurk-8. Such variants include polypeptides which are substantially homologous to native Lurk-8, but which differ from the amino acid sequence of native Lurk-8 by virtue of one or more deletions, insertions or substitutions. Likewise, the DNA encoding the Lurk-8 of the present invention comprises variants that differ from the native Lurk-8 DNA due to one or more deletions, insertions or substitutions, but which encode biologically active Lurk-8 polypeptides. As referred to in Luck-8, the term " biologically active " indicates that Luck-8 can bind a HEK or an ELK.

The modified DNA or amino acid sequence is preferably at least 80% identical, more preferably at least 90% identical to the native Lurk-8 sequence. Percentages of identification are described, for example, in Devereua et al. (Nucl. Acids Res. 12: 387, 1984), Sixth Edition] and comparing sequence information using a GAP computer available from UWGCG, University of Wisconsin. The preferred designator of the GAP program is

(1) Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. A comparison matrix of monomers for the nucleotides (containing a value of 1 for the assays and a value of 0 for the non-assays), as described in J. Biol., 353-358, 1979; : 6745, 1986), < RTI ID = 0.0 >

(2) Penalty 3.0 for each interval and in addition to each signal in each interval penalties 0.10 and

(3) No penalty during interval of terminal

.

Another example of a variety of amino acid sequences include traditional substitutions that mean that at least one amino acid residue of native Lurk-8 is replaced by a different residue, while a normally substituted Lurk-8 polypeptide retains the desired biological activity of the native protein (For example, the ability to combine with an elk or a het). Examples of traditional substitutions include substituents of residues that do not alter the secondary or tertiary structure of the protein.

A given amino acid can be replaced by a residue having similar physicochemical properties. Examples of common substituents include substituents of one aliphatic moiety for another, such as between IIe, Val, Leu, or Ala, or one substituent for another, such as between Lys and Arg, Glu and Asp, Gln and Asn Include substituents of polar residues.

Other homologous substitutions related to substituents of the entire site with similar hydrophobic properties are well known.

Moreover, the invention encompasses Lurk-8 polypeptides, either alone or in combination with a combined native-pattern glycosylation. Luck-8 expressed in a yeast or mammalian expression system (e. G., COS-7 cells) may be similar or significantly different from the glycosylation pattern where the molecular weight depends on the choice of natural Lurk-8 polypeptide and expression system . Expression of a Lurk-8 polypeptide in a bacterial expression system such as E. coli provides non-glycosylated molecules.

The N-glycosylation site may be adjusted to exclude glycosylation which results in the expression of more homogeneous and reduced carbohydrates, homologous to the mammalian and yeast expression systems. In the eukaryotic polypeptide, the N-glycosylation site is characterized by the amino acid triplet Asn-X-Y (where X is an amino acid except Pro and Y is Ser or Thr). The human lurk-8 protein of SEQ ID NO: 2 contains one triplet at amino acids 183 to 185 of SEQ ID NO: Appropriate substitutions, additions or deletions to the nucleotide sequence encoding these triplets result in the prevention of attachment of carbohydrate moieties to the Asn moiety bond. The modification of a single nucleotide is selected so that Asn is replaced by another amino acid, e. G., Sufficiently inactive at the N-glycosylation site. Known methods of inactivating N-glycosylation sites in proteins are described in U.S. Patent No. 5,071,972 and European Patent No. 276,846.

In other examples of variants, sequences encoding Cys residues that are not essential for biological activity can be modified to inhibit the formation of erroneous intramolecular disulfide bridges at the time of regeneration, thereby eliminating Cys residues or replacing them with other amino acids. Cysteine residues corresponding to the four cysteines retained in the Lack protein were found at the positions of SEQ ID NOS: 35, 65, 77 and 129. These four cysteines preferably remain unmodified in the Luck-8 variant.

In yeast systems in which KEX2 protease activity is present, other modifications are made by modification of adjacent dibasic amino acid residues to increase expression. EP 212,914 discloses the use of site specific mutagenesis to inactivate KEX2 protease processing sites in proteins. KEX2 protease processing sites are inactivated by deletion, addition or substitution of residues that modify the Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Human luc-8 contains contiguous base residue pairs at amino acids 13-14, 63-64, 151-152, 225-226, 226-227, and 227-228 of Sequence ID No. 2. Lys-Lys pair is not affected by KEX2 cleavage, and the conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a traditional and preferred approach to inactivating the KEX2 region.

Naturally occurring Lurk-8 variants are also encompassed by the present invention. An example of a variant is a protein arising from alternate mRNA junctions or from proteolytic cleavage of Lurk-8 proteins. For example, alternate splicing of mRNA produces an incised but biologically active Lurk-8 protein, such as the naturally occurring soluble form of the protein. Modifications due to protein hydrolysis include, for example, expression in different types of host cells resulting from proteolytic cleavage of one or more terminal amino acids from the Lurk-8 protein (generally from 1-5 amino acid end) Lt; RTI ID = 0.0 > N- or < / RTI > Thus, a Lurk-8 protein in which the N-terminal residue is one of the amino acids 1 to 5 of SEQ ID NO: 2 and the C-terminal residue is one of the amino acids 308 to 313 of SEQ ID NO: 2 is particularly provided herein. For soluble luc-8, the C-terminal residue may be one of amino acids 192-197 of SEQ ID NO: 2. The Lurk-8 protein, which differs from the amino acid sequence of SEQ ID NO: 2, can be attributed to the genetic polymorphism observed in the text (a variant of the allelic entity that produces the protein).

One isolated Lurk-8 cDNA contains a single nucleotide substitution compared to the cDNA described in Example 1. The sequence of the modified chain LUC-8 DNA is the DNA sequence shown in SEQ ID NO: 1 in that the nucleotide at the 1370 position of the variant is cytosine (C) rather than guanine (G) found at the position of SEQ ID NO: . The residue at position 298 in the amino acid sequence of this Lurk-8 protein is leucine.

Those skilled in the art will recognize that the above-mentioned boundaries of the region of the protein are close to discussion of the various regions of the signal peptide and the Lurk-8 protein. The boundaries of the trans membrane region (which can be predicted using a computer program sold for that purpose) are different from those described above. Thus, soluble Lurk-8 polypeptides in which the C-terminus of the extracellular domain is different from the identified residues are observed in the text. As in another example, cleavage of a signal peptide may occur at sites other than those predicted by a computer program. Moreover, it is recognized that the preparation of a protein may comprise a mixture of protein molecules with different N-terminal amino acids, which is due to the cleavage of the signal protein at one or more sites.

A computer analysis of human Lurk-8 protein indicates that the signal peptide will likely occur after amino acid-1 of SEQ ID NO: The four selective signal peptide cleavage sites predicted by the computer program are located after residues 3, -5, 2, and -2 (likely) from SEQ ID NO: 2. Thus, mature human luc-8 polypeptides wherein the N-terminal amino acid is selected from residues at positions 4, -4, 3, and -1 are provided herein, with the exception that the N-terminal amino acid is a residue at position 1.

Modifications and derivatives of the native Lurk-8 protein are produced by mutations of the nucleotide sequence encoding the native Lurk-8 polypeptide. Mutations can be introduced at specific sites by synthesis of oligonucleotides containing mutant sequences and can be flanked by restriction sites that can ligate to fragments of native sequence. In the ligations described below, the resulting rearranged sequence encodes a homologous species having the desired amino acid insertion, substitution or deletion. Alternatively, oligonucleotide-directed region-specific mutagenesis methods can be used to introduce the desired mutations. A method for achieving such a modification is 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 Natl Acad Sci USA 82: 488, 1985); Kunkel et al. (Methods in Enzymol. 154: 367, 1987); And those disclosed by U.S. Patent Nos. 4,518,584 and 4,737,462.

Luck-8 can be modified to form a luck-8 derivative by forming a covalent or cohesive bond with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups, and the like. A covalent derivative of luc-8 is prepared by linking a chemical moiety to a functional group on a Luck-8 amino acid side chain or at the N-terminal or C-terminus of a Luck-8 polypeptide or its extracellular domain. Other derivatives of Lurk-8 within the scope of the present invention include covalent or aggregation conjugates of Lurk-8 polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusion .

Fusion of Lurk-8 polypeptides may include the addition of peptides and Lurk-8 added to facilitate purification. For example, such peptides include poly-His or the peptides of the invention as described in U.S. Patent No. 5,011,912 and Hopp et al., Bio / Technology 6: 1204, 1988. One of such peptides is an antigen, a registered trademark flag peptide that provides an epitope reversibly bound by a specific monoclonal antibody that can be readily purified and rapidly tested for expression of the recombinant protein, Asp-Tyr -Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 3). As disclosed in U.S. Patent No. 5,011,912, which is hereby incorporated by reference, a murine hybridomera designated 4E11 produces a monoclonal antibody that binds with a registered trademark flag peptide in the presence of certain divalent metal cations . The 4E11 hybridoma cell line was deposited with the American Type Culture Collection under accession number HB 9259. Monoclonal antibodies that bind to the trademark flag peptide can be obtained from Eastman Kodak Company (Scientific Imaging Systems Division, New Haven, Conn.).

Lurk-8 proteins (including fragments and variants) can be tested for their ability to bind HEK or ELK in appropriate tests. The biological activity of Lurk-8 can be determined, for example, by testing the ability of a variant to compete with natural Lurk-8 binding to a heck or elk (i. E., A competitive binding test)

Competitive binding assays can be carried out using conventional systematic assays as described below. Reactants available for competitive binding assays include radiolabeled, soluble Lurk-8 and complete HEK / ELK-expressing cells. For example, radiolabeled soluble natural Lurk-8 can be used to compete with soluble Lurk-8 variants that make cell surface binding with a heck or elk. The soluble Hec / Fc or elk / Fc fusion protein bound via the interaction of the Fc portion with protein A or protein G (in the solid phase) can be replaced with a solid phase instead of the complete cell. Chromatography columns containing Protein A and Protein G include those available from the Re-Piscataway, Pharmacia Biotech, New Jersey, USA. Other types of competitive binding assays utilize radiolabeled soluble Heck or elk and complete cells expressing Lurk-8 such as HEK / Fc or elk / Fc fusion proteins. In addition, Lurk-8 can be tested for its ability to compete with one of the other Lurk proteins (above, Lurk 1-7) to bind with the elk or HEK. (Scatchard, Ann. NY: Acad. Sci. 51: 660, 1949) to obtain quantitative results, while qualitative results could be obtained by competitive autoradiographic plate binding assays Can be used.

It is possible to combine the LUCK-8 of the present invention with other prisoners of the FEF (see Background Art). Such coupling can be analyzed using suitable tests as described above.

Uses of LUCK-8 that occur in association with elk and heck include, but are not limited to: Luck-8 finds its use as a protein purification reagent. The Lurk-8 polypeptide may be attached to a solid support material and may be used to purify the Hek or elk protein by affinity chromatography. In particular, the fusion protein containing the receptor binding region of the Lurk-8 fragment or Lurk-8 in an embodiment may be attached to the solid support in the usual order. As an example, a chromatography column containing a functional group that reacts with a functional group for an amino acid side chain of a protein can be obtained (Piscataway Pharmacia Biotech, New York, USA). Lurk-8 / Fc fusion protein can be attached to protein A- or protein G- containing the chromatographic column through interaction with the Fc moiety.

Lurk-8 proteins also find use in purifying or identifying cells expressing a hack or elk on the cell surface. Luck-8 (or its fusion or fragment) should be in solid form as a column chromatography matrix or as a suitable suitable substrate. For example, magnetic microspheres can be coated with Lurk-8 and loaded in a culture vessel through a magnetic field. A suspension of cell mixtures containing heck / elk-expressing cells is contacted with a solid phase having Luck-8. Cells expressing a hack or elk at the cell surface are bound to immobilized Lurk-8 and then unbound cells are washed.

Alternatively, a mixture of cells thought to contain heck / elk + cells may be initially cultured with biotinylated Lurk-8. The incubation period is sufficient for binding with the heck / elk and typically over an hour. The resulting mixture is then passed through a column filled with avidin coated beads, thus providing for binding of the cells to the bead due to the high affinity of biotin for avidin. The order of using avidin-coated beads is described in Berenson, et al., J. Cell. Biochem., 10D: 239, 1986]. The washing of unbound material and the release of bound cells are carried out using conventional methods.

A variety of in vitro studies or in vivo methods can then be used to relocate the purified cell populations, for example, to tissues of mammals. For clarification, neutrophil cells expressing the elk can be isolated by the above methods and administered to mammals with neurodegenerative disorders. Heck ⁺ cells contain certain lucemia cells (identified below). Isolated lucerne cells are used, for example, in studies on the effects of various drugs on cells.

In order to identify additional types of cells expressing a hack or elk on the cell surface, Lurk-8 may be associated with a detectable moiety, such as a radionuclide. As one example, it is a radiolabeled with ¹²⁵ I can be performed by one of several standard phylogenetic classification for generating a high specificity in the functional group-labeled with ¹²⁵ I activity Te -8 molecule. Other detectable moieties include enzymes capable of catalyzing colorimetric or fluorescent colorimetric reactions. Cells that test for HEK / ELK-expression are contacted with labeled LUC-8. After incubation, unbound label luc-8 is removed and the presence or absence of a detectable moiety on the cell is measured.

The Lurk-8 protein has also been found to measure the biological activity of an elk or HEK protein in view of the binding affinity with Lurk-8. Thus, the Lurk-8 protein can be used, for example, to conduct "quality assurance" studies to monitor the stability and shelf life of elk or heck proteins under different conditions. 8 can be used for binding affinity studies for the measurement of the biological activity of elk proteins stored at different temperatures for the purposes of illustration and where different cell types are produced. Luck-8 may also be determined depending on whether biological activity is maintained after modification of the elk or HEK protein (e.g., chemical modification, incision, mutation, etc.). The binding affinity of the modified elk protein for Luck-8 is compared to the binding affinity of the unmodified elk protein to find the adverse effect of modification on the biological activity of the elk. Thus, the biological activity of the HEK protein can be assessed using LUCK-8. Thus, the biological activity of elk or heck proteins can be ascertained, for example, before being used in research studies.

Lurk-8 polypeptides have also been found for use as carriers for releasers attached to cells with receptors on the surface of elk or heck cells. Expression of HEK antigens has been reported for Lucerne cell lines, including human T-cell lucemia cell lines designated JM and HSB-2, and human pre-B cell lucemia cell lines expressing LK63 (Wicks et al. Proc. Natl. Acad. Sci. USA, 89: 1611, 1992; Boyd et al., J. Biol. Chem. 267: 3262, 1992). Thus, the Lurk-8 protein can be used to deliver a diagnostic or therapeutic agent in vivo or in an in vitro method to these cells (or other cell types in which the expression of a hack or elk on the cell surface is found).

One example of such a use is to expose the Heck ⁺ cell line to the Therapeutic / Lurk-8 complex to assess whether the treatment exhibits cytotoxicity towards Lucemia cells. Many different therapeutic agents attached to Lurk-8 can be included in a test to compare and detect the cytotoxic effect of agonists on Lucemia cells. The luck-8 / diagnostic agent complex can be used to detect the presence of HEK ⁺ in vivo or ex vivo.

Detectable (diagnostic) therapeutic agents that can be attached to the Lurk-8 polypeptide include drugs, toxins, radionuclides, chromophores, enzymes that catalyze the reaction of a colorimetric or fluorescent color, etc., with specific agonists selected according to the intended use, It is not limited. Examples of drugs include, for example, nitrogen mustard such as L-phenylalanine nitrogen mustard or cyclophosphamide, an intercalating agent such as cisdiaminodichloroplatinum, antimetabolites such as 5-fluorouracil, vinca alkaloids such as vincristine , Antiinflammatory agents, antiinflammatory agents, antiinflammatory agents, antiinflammatory agents, antiinflammatory agents, antiinflammatory agents, antiinflammatory agents,

The toxins include fungal toxins such as lysine, epinephrine, diphtheria toxin, pseudomonas aruginotoxin A, ribosomal inactivating protein, tricothecene, and derivatives and fragments thereof (e.g., single chain). Suitable radioactive nucleotides for diagnostic purposes include, but are not limited to, ¹²³ I, ¹³¹ I, ^99m Tc, ¹¹¹ In, and ⁷⁶ Br. Suitable radionuclides for treatment include, but are not limited to, ¹³¹ I, ²¹¹ At, ⁷⁷ Br, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, and ⁶⁷ Cu.

Such agents may be attached to Lurk-8 by suitable conventional methods. Lurk-8, which is a protein, contains a functional group on the amino acid side chain that can react with a functional group on a desired agonist, for example, to form a covalent bond. Alternatively, the protein or agent can be derivatized to generate or attach the desired reactive functional group. The derivatization may involve attachment of one of the bi-functional coupling agents capable of attaching various molecules to the protein (Pierce Chemical Company, Rocjford, Illinois). A variety of techniques for radioactively labeled proteins are known. The radionuclide metal may be attached to Luck-8 using, for example, a suitable bifunctional chelating agent.

Thus, complexes containing Lurk-8 and suitable diagnostic or therapeutic agents (preferably covalently bound) have been prepared. The complex may be administered or used in an amount suitable for the particular use.

Another use of Lurk-8 of the present invention is in its use as a research tool to study the role that Lurk-8 may play in the differentiation or growth of cells with elk or hek receptors in relation to elk or heck. The Lurk-8 polypeptides of the invention may also be used in in vitro tests for their interaction or detection of Lurk-8 or elk. Likewise, Lurk-8 found its use in the interaction of Hek and Luck-8 or in tests for Hek. It is suggested that Hek may play a role in tumorigenesis (Boyd et al., Supra)

The polypeptides and Lurk-8 DNA of the present invention may be useful in the development of a disorder mediated (either directly or indirectly) by an incomplete or insufficient amount of LUC-8. Lurk-8 polypeptides may be administered to such disordered mammals. Alternatively, a gene therapy approach may be used. It is possible to detect incomplete Lurk-8 gene and rearrange it with a gene which makes general Luck-8-encoding according to the contents disclosed in the text of the natural Lurk-8 nucleotide sequence. Infective genes can be detected in in vitro diagnostic tests, and natural-luc-8 nucleotide sequences compared to the Lurk-8 gene obtained from humans suspected of having defects in this gene are disclosed herein. As discussed above, when various mouse tissues were analyzed for elk mRNA, transcription was found only in the brain and testes (Lhotak et al., Supra). Through the expression of receptors for the luc protein in the nervous tissue, it becomes possible to study the role of the luc protein in the regeneration or development of the nervous system. Lurk-7 has been implicated in axon guidance and axon formation (Winslow et al., Neuron 14: 973-981, 1995; Drescher et al., Cell 82: 359-370, 1995). The Lurk-8 of the present invention can be used to study the effects of binding of Lurk-8 to receptors on neutral tissues. Luck-8 can investigate its role in inducing or regulating processes associated with the nervous system. Luck-8 may be administered in vivo to facilitate or modulate the development of the nervous system.

It has been reported that some of the lurk proteins have neuroprotective properties, for example, to protect the hippocampal neurons against glutamate-mediated excitotoxicity. The involvement of excitatory elements has been established in many disorders of the nervous system. The response to glutamate is a common function in the developmental light maturation of the central nervous system (CNS). However, in addition to its general role in excitatory synaptic transmission and plasticity, glutamate also has a number of CNS, including but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, seizures (ischemia), epilepsy and AIDS- (Lution et al., Neuron 7: 111, 1991; and Andersson et al., 1999). al., Eur., J. Neurosci., 3:66, 1991).

The Lurk-8 polypeptides provided herein find use in methods of treating disorders associated with nerve tissue in contact with Lurk-8. Such disorders include disorders of the nervous system or disorders of the chronic or acute nervous system. However, examples of disorders include, but are not limited to, the above conditions associated with dysfunction of the CNS. Luck-8 can be administered to mammals, including humans, that affect such conditions.

It has been found that some of the Lurk proteins promote angiogenesis. Likewise, it can be found that LUC-8 stimulates new blood vessel formation in the transplanted tissue and promotes angiogenesis that may be useful for wound healing, or treatment under conditions where the desired angiogenesis is increased. Compositions comprising an effective amount of a Lurk-8 polypeptide of the invention in association with other components such as physiologically acceptable diluents, carriers or excipients are provided herein. Luck-8 may be formulated according to known methods used to prepare pharmaceutically useful compositions. Luck-8 may be formulated with pharmaceutically acceptable excipients such as saline, tris-hydrochloric acid, acetate and phosphate buffer solutions, preservatives such as thimerosal, benzyl alcohol, parabens, emulsifiers, solubilizers, Can be combined with the carrier as the sole active substance or in combination with other known active substances. Suitable formulations for pharmaceutical compositions are described in Remington ' s Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA).

Moreover, such compositions may be formulated with a wide range of excipients such as polyethylene glycol (PEG), complexed with metal ions or incorporated into polymeric compounds such as polyacetic acid, polylactic acid, hydrogel, dextran, Or a multilamellar vacuole, cyclic red blood cells, or Lurk-8 incorporated into the blast cells. Such compositions will affect the physical state, solubility, stability, rate of in vivo release and rate of in vivo clearance of LUC-8, and will be selected according to the intended use. Luck-8 may also be conjugated to a tissue-specific receptor, a ligand, or an antibody that couples a ligand of an antigen or a tissue-specific receptor. In addition, the use of Lurk-8 expressed on the cell surface can be found.

Such compositions may contain the Lurk-8 polypeptides in any of the forms described herein, such as natural proteins, modifications, derivatives, oligomers and biologically active fragments. In one embodiment, the composition contains an oligomer comprising a soluble Lurk-8 polypeptide, preferably a soluble Lurk-8 polypeptide.

Luck-8 may be administered, for example, locally, intracervically, or by inhalation in a suitable manner. The term " extravasary " includes, for example, injection by subcutaneous, intravenous, or intramuscular routes, including topical administration of the disease or injured site. Continuous release from the insert also appears. Those skilled in the art will recognize that suitable dosages will vary depending upon such factors as the nature of the disorder to be treated, the body weight, age and general condition of the patient, and the route of administration. A primary dose can be determined according to animal testing, and the measurement of the dose for human administration is performed by a technically acceptable method.

Oligomeric Form of Luck-8

Included in the present invention are oligomers containing Lurk-8 polypeptides. Luck-8 oligomers may be present in the form of covalently or non-covalently bonded dimers, trimer or higher oligomers.

One of the present inventions relates to an oligomer comprising a multiple luc-8 polypeptide bound via a covalent or noncovalent interaction between peptide moieties fused to a luc-8 polypeptide. Such peptides may be peptide linkers (spacers), or peptides with properties that promote oligomerization. Some of the polypeptides obtained from the leucine zipper and antibodies are present between peptides capable of promoting oligomerization of the attached Lurk-8 polypeptide, as described in more detail below.

In particular, in an embodiment, the oligomer comprises from two to four Lurk-8 polypeptides. The Luck-8 portion of the oligomer may be a soluble polypeptide, as described above.

In one alternative, the Luck-8 oligomer is prepared using a polypeptide obtained from an immunoglobulin. The preparation of a fusion protein comprising a somewhat heterogeneous polypeptide fused to various regions of an antibody-derived polypeptide (including the Fc region) is described, for example, in Ashkenazi et al., PNAS USA 88: 10535, 1991 ); Byrn et al. (Nature 344: 677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992).

One preferred example of the present invention relates to a luc-8 dimer comprising two fusion proteins made by fusion of Lurk-8 to the Fc region of the antibody. Preferably the Fc polypeptide is fused to the C-terminus of soluble Lurk-8. Genetic fusion encoding the Lurk-8 / Fc fusion protein can be inserted into an appropriate expression vector. The Lurk-8 / Fc fusion protein is expressed in host cells transformed with recombinant expression vectors and is allowed to aggregate with antibody molecules that are internally linked in disulfide bond form between the Fc portions producing bivalent Lurk8 .

Provided herein is a fusion protein comprising a Lurk-8 polypeptide that fuses an Fc polypeptide obtained from an antibody. A dimer containing two fusion proteins bound through disulfide bonds between the Fc portions and DNA encoding such fusion proteins are also provided. The term " Fc polypeptide " as used herein includes native and mutant forms of the polypeptide obtained from the Fc region of an antibody. Also included are dissected forms of the polypeptide containing a hinge region that facilitates dimerization. One suitable Fc polypeptide described in PCT application WO 93/10151 is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of human IgG1 antibody. Other useful Fc polypeptides are the Fc mutations disclosed in U.S. Patent No. 5,457,035 and Baum et al., (EMBO J. 13: 3992-4001, 1994). The amino acid sequence of this mutant is identical to the native Fc sequence shown in WO 93/10151 except that the amino acid 19 was changed from Leu to Ala, the amino acid 20 was changed from Leu to Glu, and the amino acid 22 was changed from Gly to Als. Mutations represent reduced affinity for Fc consumers.

In another embodiment, Luck-8 can be substituted with various regions of the light chain or heavy chain of the antibody. When the fusion protein is made up of heavy chains and light chains of the antibody it is possible to form a Luck-8 oligomer such as the four Lurk-8 extracellular domains.

The oligomer is also a fusion protein comprising a multi-Luck-8 polypeptide with a peptide linker (spacer peptide) or without a peptide linker. Suitable peptidylinkers include those described in U.S. Patent Nos. 4,751,180 and 4,935,233, which are incorporated herein by reference. DNA sequences encoding the desired peptide linker can be inserted into or between the same reading frame as in DNA encoding Lurk-8 using appropriate conventional techniques. For example, a chemically synthesized oligonucleotide encoding a linker can be ligated between sequences encoding Lurk-8. In an embodiment, the fusion protein comprises two to four soluble Lurk-8 polypeptides separated by a peptide linker.

Another manufacturing method of oligomeric Luck-8 relates to the use of Louis Xinzip. The lysine zipper region is a protein that promotes oligomerization of the protein found. Lewis zipper was originally identified in several DNA-binding proteins (Landschulz et al., Science 240: 1759, 1988), since many different proteins were originally discovered. Among the known ruby zippers are the naturally occurring derivatives and peptides that are dimerized or trimerized. Examples of suitable leucine zipper regions for preparing soluble oligomeric proteins are described in PCT application WO 94/10308 and are described in Hoppe et al. (FEBS Letters 344: 191, 1994) and U.S. Patent Application Serial No. 08 / 446,922 Are thus included in the reference. [0035] The term " liposin " A recombinant fusion protein comprising a soluble luc-8 polypeptide fused to a lucine zipper peptide is expressed in a suitable host cell and soluble oligomeric luc-8 in which the form is recovered from the culture supernatant.

Oligomeric Luck-8 possesses the properties of 2, 3, etc., which combine with the location of the elk or heck. The fusion protein (and the oligomer formed therefrom) containing the Fc portion provides the advantage of easy purification by affinity chromatography on protein A or protein G columns.

Expression system

Suitable host cells for expression of Lurk-8 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Suitable cloning and expression vectors used with bacterial, fungal, yeast and mammalian cytoplasmic hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems can also be used to generate Lurk-8 polypeptides using RNA obtained from the DNA constructs disclosed herein. The expression vector may comprise a signal fused to the N-terminus of the Lurk-8 polypeptide or DNA encoding the leader peptide. Signal or leader peptide to translate Luck-8 from its synthetic site to the outer surface or inner surface of the cell wall or cell membrane. The signal or leader peptide is cleaved from the mature L-8 polypeptide. The signal or leader peptide is cleaved from the mature L-8 polypeptide. The choice of signal or leader peptide will depend on the type of host cell to be used.

Suitable prokaryotic host cells for transformation include, for example, various species in E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces, and grapevines. In a prokaryotic host cell such as E. coli, the Lurk-8 polypeptide comprises an N-terminal methionine residue that facilitates the expression of the recombinant polypeptide in the host cell of the prokaryotic cell. The N-terminal Met can be cleaved from the expressed recombinant Lurk-8 polypeptide.

The Lurk-8 polypeptide may be expressed from a yeast host cell, preferably from a yeast cell line (e. G., Brewer yeast). Fitchia, Kay. Other genus of yeast such as lactis, or clobberomyces may also be used. The yeast vector may contain the origin of the replication sequence, the autonomous replication sequence (ARS), the promoter region, the polyadenylation sequence, the transcription termination sequence and the optional marker gene from the 2μ yeast plasmid.

Suitable promoter sequences for yeast vectors include, but are not limited to, enolase, glyceraldehyde-3-phosphate, dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, Among others, such as clicellite mutease, pyruvate kinase, triocatephosphate isomerase, phosphoglucose isomerase and glucokinase, metallothionine, 3-phosphoclorecylline kinase (Hitzeman et al., J 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978), as well as other suitable enzymes (Hess et al., J. Adv. Enzyme Reg. do. See Russell et < RTI ID = 0.0 > al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Other vectors and promoters suitable for use in yeast expression are further described in Fleer et. al., Gene, 107: 285-195 (1991); And van den Berg et. al., Bio / Technology, 8: 135-139 (1990).

The shuttle vector replicable in yeast and Escherichia coli can be made by inserting the DNA sequence from pRB322 into the desired yeast vector for selection and replication of E. coli (Amp ^r gene and origin of replication).

A suitable leader sequence (e. G., An alpha-factor leader in the yeast group) may be used directly in the secretin of a L-8 polypeptide from yeast cells. The? -finger leader sequence is generally inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30: 933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330, 1984; U.S. Patent No. 4,546,082 and EP 324,274. Other leader sequences suitable for promoting secretin of a recombinant polypeptide from a yeast host are known in the art. The leader sequence may be modified in the vicinity of the 3 'end containing one or more restriction sites. This will facilitate fusion of the leader sequence of the structural gene. The protocol of yeast transformation is known to those skilled in the art. One such protocol is described in Hinnen et al., Proc. natl. Acad. Sci. USA 75: 1929, 1978.). The protocol, such as the Hinne, selects Trp ⁺ in selective media consisting of 0.67% yeast nitrogen-based, 0.5% casamino acid, 2% glucose, 10 ug / ml adenine and 20 ug / ml uracil.

Yeast host cells transformed by a vector containing an ADH2 promoter sequence may be grown to induce expression in an " enriched " medium. Examples of abundant media consist of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ug / ml adenine and 80 ug / ml uracil. Suppression of the ADH2 promoter occurs when glucose is consumed from the medium.

Host cell culture systems for mammals and insects may also be used to express recombinant Lurk-8 polypeptides. The Baku virus system for producing heterogeneous proteins in insect cells has been reviewed by Luckow and Summers, Bio / Technology 6:47 (1988). A constructed cell line of mammalian origin was also used. Examples of suitable mammalian host cell lines are described in McMahan et al. (ATCC CRL 1651; Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells, and the like, as described in EMBO J. 10: 2821,1991 (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines and African green monkey ovarian cell line CV1 (ATCC CCL 70), ovarian CV-1 / EBNA-1 cell line ATCC CRL 10478).

Transcriptional and translational control sequences of mammalian host cell expression vectors can be ablated from the viral genome. Commonly used promoter sequences and enhancer sequences are obtained from poliomavirus, anenovirus 2, simian virus 40 (SV40), and human cell-spreading viruses. DNA sequences obtained from the SV40 viral genome may be used, for example, to provide SV40, early and late promoters, enhancers, splices and polyadenosine sites to provide other genetic elements for the expression of structural gene sequences in mammalian host cells . Early and late viruses are particularly useful because they can be easily obtained from the viral genome as fragments that can contain the viral origin of replication (Fiers et al., Nature 273: 113, 1978).

Smaller or larger SV40 fragments may also be used and provide an approximately 250 bp sequence extending from Hind III towards the Bgl I site located at the SV40 virus origin of the replication site.

Examples of expression vectors used in mammalian host cells are those prepared as disclosed in Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983). Useful epithelial cells are described by Cosman et al. (Mol. Immunol. 23: 935, 1986). A very useful expression vector, PMLSV N1 / N4, as described in Cosman et al., Nature 312: 768, 1984, was deposited as ATCC 39890. Other expression vectors suitable for use in mammalian host cells include pDC201 (Sims et al., Science 241: 585, 1988), pDC302 (Mosley et al., Cell, 59: 335, 1989), pDC406 (Mcmahan et al. EMBO J. 10: 2821, 1991), HAV-EO (Dower et al., J. Immunol. 142: 4314, 1989), and EP-A-0367566 and WO 91/18982. Alternatively, the vector obtained from the retrobiose can be selected.

In the place of the natural signal sequence, the signal sequence of IL-7 described in U.S. Patent No. 4,965,195, the signal sequence of IL-2 described in Cosman et al., Nanure 312: 768 (1984) Heterologous signal sequences may be added, such as IL-4 receptor signal peptides, Type I IL-1 receptor signal peptides as described in U.S. Pat. No. 4,968,607, and Type II IL-1 receptor signal peptides as described in EP 460,846.

Luck-8 protein purification

The Lurk-8 polypeptides of the present invention can be produced by recombinant expression systems described above or can be purified from naturally occurring cells. One method of producing Lurk-8 involves culturing host cells transformed with an expression vector comprising a DNA sequence encoding Lurk-8 under conditions sufficient to promote expression of Lurk-8. Next, depending on the expression system used and whether LUC-8 is secreted from the cells, LUC-8 is recovered from the culture medium or cell extracts. In one embodiment, the human Lurk-8 protein comprises the amino acid sequence of a protein expressed by a host cell transformed with an expression vector containing the Lurk-8 cDNA found in strain ATCC 97441.

As is known to those of skill in the art, the purification sequence of the recombinant protein will vary depending on factors such as the type of host cell used and whether the recombinant protein is secreted into the culture medium. Other types of contaminants to be removed that may vary depending upon the particular host cell used to express the desired protein are considered.

For example, if an expression system that secretes recombinant protein is used, the culture medium may first be concentrated using a protein concentration filter (e. G., Amicon or Millipore pellicron sold, ultrafiltration units). The concentrate, concentrate, described below, may be used in purification matrices such as gel filtration matrices. Also, an anion exchange resin, for example, a matrix or a substrate having a diethylaminoethyl (DEAE) group can be used. The matrix may be acrylamide, azarose, dextran, cellulose or other support materials commonly used in protein purification. A cation exchange step can also be used. Suitable cation exchangers may include several insoluble matrices including sulfopropyl or carboxymethyl groups. Sulfopropyl group is preferred. Furthermore, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC medium (eg silica gel with methyl or other aliphatic substituents) can be used. Some or all of the purification steps described below may be used to provide a purified Lurk-8 protein.

An additional option is an affinity chromatographic paper using chromatography containing heck, elk or antibody binding to Luck-8. Luck-8 polypeptides can be recovered from the affinity column using conventional techniques (elution of high salt buffer) and then dialyzed with a buffer of low salt for use.

Recombinant proteins made in bacterial media can be isolated by initial cleavage of host cells, centrifugation, extraction from cell pellets if it is an insoluble polypeptide, extraction from supernatant fluids if soluble polypeptides, followed by one or more centrifugation Separation, salting out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be used in the final purification step. Microviral cells can be cleaved by conventional methods, including freeze-thaw cycling, sonolysis, mechanical cleavage, or the use of cell homogenates.

In yeast host cells, Lurk-8 is preferably expressed as secreted polypeptides to simplify purification. Recombinant polypeptides secreted from yeast host cells are described in Urdal et al. (J. Chromatog. 296: 171, 1984). This document discloses two successive reversed-phase HPLC steps for the purification of recombinant human IL-2 on a preparative HPLC column.

The desired purity depends on the intended use of the protein. Relatively high purity is desirable when the protein is administered, for example, in vivo. Advantageously, the Lurk-8 polypeptide is purified so that the protein mass corresponding to the other (non-Luke-8) protein can be detected by analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the art will appreciate that due to the unusual glycosylation, specific post-translational processing, and the like, multigens corresponding to the Lurk-8 protein can be visualized by SDS-PAGE. Most preferably, as indicated in the single protein band on the analysis by SDS-PAGE, Luck-8 is substantially purified to homogeneity. The protein band can be visualized by staining with silver, blue staining with coma or by automatic radiography (if the protein is radio labeled).

Nucleic acid and its uses

As described above, the present invention provides isolated luc-8 nucleic acids useful in the production of Lurk-8 polypeptides. Such nucleic acids include, but are not limited to, the human Lurk-8 DNA of SEQ ID NO: 1 in the same-day and double-stranded form as well as the RNA replenishment thereof. The Lurk-8 DNA of the present invention includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and recombinant type thereof. The genomic DNA can be isolated as a probe by conventional techniques using the cDNA isolated in Example 1, or a suitable fragment thereof.

Preferred examples of DNA encoding Lurk-8 include nucleotides 398 to 1420 of SEQ ID NO: 1 (which encodes a human Lurk-8 full length comprising an N-terminal single peptide) and nucleotide 479 of SEQ ID NO: To 1420 (which encodes the entire mature human Lurk-8). A preferred embodiment of DNA encoding soluble human luc-8 comprises nucleotides 398 to 1069 (single peptide and extracellular region) of SEQ ID NO: 1 and nucleotides 479 to 1069 of SEQ ID NO: 1 ).

The present invention further provides fragments of the Lurk-8 nucleotide sequence. Such fragments preferably comprise about 17 contiguous nucleotides of the Lurk-8 DNA sequence, such as, for example, 17 or more contiguous nucleotides of the human Lurk-8 sequence present in SEQ ID NO: . DNA and RNA replicants of such fragments are provided in the text in both single-stranded and double-stranded forms of Lurk-8 DNA.

Among the uses of the luc-8 nucleic acid fragments there are applications as probes. Such probes can be used in cross-species hybridization methods to isolate Lurk-8 DNA from additional mammalian species. As an example, a probe corresponding to the extracellular domain of Lurk-8 can be used. The probe also finds use in Northern and Sourthern blots to detect the presence of luc-8 nucleic acids in the above procedure and in vitro tests. The cell type of the expressing Lurk-8 can be identified. Such methods are different, depending on the intended use, in particular, and can be selected by the skilled person if the probe is of a suitable length. In a preferred embodiment, the Lurk-8 nucleic acid molecule comprises more than 30 consecutive nucleotides of the DNA sequence of SEQ ID NO: 1 and its DNA and RNA replicants. The probe may be labeled by conventional techniques (for example, ³² P)

Lurk-8 nucleic acid fragments also find use in primers, e. G., Polymerase chain reaction (PCR). The 5 ' and 3 ' primers (dP, DNA encoding soluble Lurk-8) corresponding to the end of the desired Lurk-8 DNA are used to isolate and amplify DNA using conventional PCR techniques.

Other useful fragments of the luc-8 nucleic acid include an inverted sense comprising a single-stranded nucleic acid sequence (RNA or DNA) capable of binding to target Lurk-8 mRNA (sense) or Lurk-8 DNA Or sense oligonucleotides.

According to the present invention, the inverse sense or sense oligonucleotide contains a fragment of the coding portion of the Lurk-8 cDNA. Such fragments generally comprise about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to obtain reverse sense or sense oligonucleotides based on a cDNA sequence encoding a given protein is described, for example, in Stein and Cohen (Cancer Res. 48: 2659, 1998) and van der Krol et al. (BioTechniques 6: 958, 1988).

Reversing or binding of the sense oligonucleotide to the desired nucleic acid sequence may be accomplished by blocking the transcription or translation of the sequence of interest by one of several methods including, but not limited to, axial amplification of double strands, premature termination of transcription or translation, Resulting in the formation of double strands. Thus, the inverse sense oligonucleotide can be used to block the expression of the Lurk-8 protein. In addition, the reverse sense or sense oligonucleotides are oligonucleotides having a modified sugar-phosphodiester base structure (or other sugar bonds such as those described in WO 91/06629) in which a single bond is resistant to endogenous nuclease Lt; / RTI > Oligonucleotides with binding specificity are stable in vivo (resistant to enzymatic fusions), but retain sequence specificity that can bind the nucleotide sequence for purposes.

Other embodiments of sensory or salt sense oligonucleotides include organic moieties such as those described in WO 90/10448, other moieties that increase the affinity of oligonucleotides for the desired nucleic acid sequence, such as poly- (L-lysine) Lt; RTI ID = 0.0 > oligonucleotides < / RTI > Furthermore, an ellipticine and an intercalating agent such as an alkylating agent or a metal complex can be bound to a sensory or inverse oligonucleotide in order to modify the reverse specificity for the desired nucleotide sequence or the binding specificity of the sensory oligonucleotide.

Reverse sense or oligonucleotide sense nucleotides are, for example, CaPO ₄ - a gene transfer vector, such as a bar virus (Epstein-Barr Virus) - mediated DNA transfection, by gene transfer methods including electrophoresis, or Epstein Can be introduced into cells containing the desired nucleic acid sequence. In the preferred method, the reverse sense or sense oligonucleotide is inserted into a suitable retrovirus. Cells containing the desired nucleic acid sequence are contacted with a recombinant retroviral vector in vivo or ex vivo. Suitable retroviruses include, but are not limited to, those derived from the murine leukemia M-MuLV, N2 (a retrovirus obtained from M-MuLV) or a duplex vector denoted DCT5A, DCT5B and DCT5C (WO 90/13641) Do not.

Sense or reverse sense oligonucleotides are introduced into cells containing the desired nucleic acid sequence by conjugate formation with a ligand binding molecule as disclosed in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines or other ligands that bind to cell surface receptors. Preferably the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to the corresponding molecule or receptor or to block the introduction of a variant or sensory or reverse sense oligonucleotide conjugated to the cell Do not.

Also, as described in WO / 90/10448, sense or inverse oligonucleotides can be introduced into cells containing the desired nucleic acid sequence by the formation of oligonucleotide-lipid complexes. This sensory or inverse sensory oligonucleotide-lipid complex is preferably associated with cells by endogenous lipase.

Antibody

The present invention provides antibodies that are immunoreactive with Lurk-8 polypeptides. Such antibodies specifically bind to Lurk-8 via the antigen-binding portion of the antibody (as opposed to non-antibody binding).

The polyclonal and monoclonal antibodies can be prepared by conventional methods. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Kennet et al. (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). The generation of monoclonal antibodies to Luck-8 will be further described in Example 3. [

Antigen-binding fragments of antibodies that can be generated by conventional techniques are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab, F (ab ') 2 and F (ab') ₂ fragments. Antibody fragments or derivatives produced by genetic engineering techniques are also provided.

The monoclonal antibody of the present invention includes, for example, a human chimeric antibody, which is a modification to a human body of a mouse monoclonal antibody. Such humanized antibodies can be made by conventional techniques and offer the advantage of reducing immunogenicity when administered to humans. In one alternative embodiment, a human monoclonal antibody comprises various regions of a murine antibody (or antigen binding portion thereof) and a control region obtained from a human antibody. In addition, fragments of antibodies applied to humans can include antigen binding sites of murine monoclonal antibodies and fragments of various regions obtained from human antibodies (lack of antigen binding sites). Methods for the preparation of chimeric and further processed monoclonal antibodies are described in Riechmann et al. (Nanure 332: 323, 1988), Liu et al. (PNAS 84: 3439, 1987), Larrick et al. (Bio / Technology 7: 934, 1989), and Winter and harris (TIPS 14: 139, May, 1993).

The use of antibodies is intended to test the presence of Lurk-8 polypeptides in vivo or ex vivo. Antibodies can also be used to purify Lurk-8 protein by genetic affinity chromatography.

In addition, antibodies capable of blocking the binding of Lurk-8 to receptors (e. G., Elk or heck) may be used to inhibit the tagged physiological activity by binding Lurk-8 to the receptor. Such antibodies may be used in vivo methods or may be injected into the body to inhibit Lurk-8 mediated physiological activity. Thus, it is possible to treat worsened or mediated diseases (directly or indirectly) by binding of Lurk-8 to cell surface receptors.

The present invention provides a pharmaceutical composition comprising Lurk-8 and an antibody directed to a suitable diluent, excipient or carrier. A suitable composition of such an antibody is as described above with a composition containing a Lurk-8 protein.

The invention also provides a conjugate comprising a detectable (e.g., diagnostically) therapeutic agent in combination with an antibody directed against Lurk-8. Examples of such agents are described above. The conjugates find use in vivo or in vitro methods.

The following examples are provided to further illustrate the present invention and are not to be construed as limiting the scope of the present invention.

≪ Example 1: Cloning of human Lurk8 cDNA >

CDNA encoding human Lurk-8 of the present invention was isolated by the following method. EST (Accession No. H10006) showing important homology was identified by searching the sequence data bank (tfasta) of the gene bank using the amino acid sequences of Luke-2 and Luke-5 as search terms. Using the leading frames of Luck-2 and 5 as a guide (to adjust the cleavage and stop codon resulting from insertion, deletion in EST compared to the Luck-2 and Luck-5 sequences) It became clear. This translational arrangement of ESTs with the Luke-2 and Luke-5 amino acid sequences shows about 50% sequence identity in both the Luke-2 and Luke-5 overlapped regions. The second, third, and fourth cysteines retained in Lurk-1-7 were identified during EST translation.

EST-based oligonucleotides were synthesized in polymerase chain reaction (PCR) for use as 5 ' and 3 ' primers. The prime is defined as the end of the 110 bp internal fragment of the EST. DNA from human cDNA library in phage lambda vector was used as template in PCR. DNA fragments of the expected size (110 bp) were extended from three cDNA libraries obtained from fetal brain, skin fibroblasts and pancreatic cancer.

The same two oligonucleotides used as primers in PCR were end-labeled with ³² P for use as probes. The fibroblast cDNA library of human skin was screened by hybridization at 63 degrees Celsius, followed by washing at 1 x SSC, 63 degrees Celsius. One of the hybridizing clones isolated the indicated lambda. The coding portion of this clone corresponds to the nucleotides 500 to 1420 of SEQ ID NO: 1 encoding the amino acids 8 to 313 of SEQ ID NO:

The fragment of this clone was amplified by PCR, labeled and used as a probe to screen the human fetal no cDNA library (hybridization at 63 degrees Celsius followed by 1 x SSC, washing at 63 degrees Celsius). Three hybridizing clones were isolated and the DNA sequence determined. One clone denoted by lambda 2 contained the entire length of the encrypted region.

The nucleotide sequence of the human Lurk-8 cDNA of clone lambda 2 and the corresponding amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The second human lurk-8 protein of SEQ ID NO: 2 is the N-terminal signal peptide (amino acid -27 to -1), the extracellular domain (amino acids 1 to 197), the trans-membrane (amino acids 198 to 224) Region (amino acids 225 to 313).

A sample of the cell lysate of the recombinant phage vector (human Lurk-8 cDNA λgt10 inserted into the EcoRI restriction site of the vector) was deposited with the American Type Culture Collection, Rockville, MD, USA. The sample was deposited on February 14, 1996 under the Budapest Treaty, and assigned as deposit number ATCC97441.

≪ Example 2: Binding study >

The linkage of Luck-8 to the elk or heck can be evaluated by conventional bonding tests.

Encoded amino acid sequences for DNA and murine elk cDNA are described in Lhotak et al. (Mol. Cell. Biol. 11: 2496, 1991), which is hereby incorporated by reference. The rat elk protein has a 538 amino acid extracellular domain, a 25 amino acid trans-membrane domain, and a 419 amino acid cytoplasmic domain.

Encoded amino acid sequences for DNA and murine Heck cDNA are described in Wicks et al. (Proc. Natl. Acad. Sci. USA, 89: 1611, 1992), and is hereby incorporated herein by reference. The HEK protein has a 521 amino acid extracellular domain (from the N-terminus to the C-terminus), a 24 amino acid trans-membrane domain, and a 418 amino acid cytoplasmic domain.

Recombinant soluble Elk / Fc and HEK / Fc fusion proteins are prepared by suitable methods, for example as described in PCT application WO 96/01839, and are listed herein as references. Elk / Fc and HEK / Fc fusion proteins are purified by affinity chromatography using a Protein A Sepharose column.

Cells expressing recombinant Lurk-8 on the cell surface were prepared. Luck-8 DNA could be extended by PCR. The primer used in the PCR is selected to represent the end of the coding portion of the Lurk-8 DNA, and the Xho I restriction site at the 5 'end and the Not I site at the 3' end of the extended DNA are added.

The PCR reaction product is digested with Xho I and Not I and inserted into an expression vector which is cleaved with Sal I (compatible with Xho I). The expression vector designated pDC410 is also a vector of mammal that replicates in E. coli and is similar to pDC406 (McMahan et al., EMBO J. 10: 2821, 1991).

The pDC410 multiple cloning site (mcs) differs from pDC406 in that it contains an additional restriction and three stop codons (one of each of the leading frames). The lower part of the T7 polymerase promoter of mcs allows the DNA sequence to be easily inserted into mcs. Moreover, the EBV origin of replication is replaced by DNA encoding SV40 to T antigen in pDC410 (obtained from the SV40 promoter).

CV-1-EBNA-1 cells in a 10 cm ² dish are transfected with a recombinant expression vector containing Lurk-8 DNA. The CV-1-EBNA-1 cell line (ATCC CRL 10478) structurally expresses EBV nuclear antigen-1 from the CMV immediate early enhancer / promoter. CV-1-EBNA-1 cells are described in McMahan et al. (ATCC CCL 70) from African green monkey kidney cell line CV-1 (EMBO J. 10: 2821, 1991).

Transfected cells are cultured for 24 hours and the cells in each dish are split into 24-well plates. After incubation for an additional 48 hours, the transfected cells were incubated with binding medium (about 4 x 10 ⁴ cells / well) containing 25 milligrams / milliliter of bovine serum albumin, 2 milligrams / milliliter of sodium azide, RPMI), BM-NFDM, and added to 50 milligrams / milliliter of non-fat dry milk. The cells are then incubated at various concentrations of the elk / Fc fusion protein or the HEK / Fc protein fusion protein at 37 degrees Celsius for 1 hour. Next, the cells were washed and cultured constantly while saturating the concentration of ¹²⁵ I-mouse anti-human IgG in the binding medium while gently stirring at 37 ° C for 1 hour. After washing as a whole, the cells were released via trypsinization.

The used mouse anti-human IgG is directed against the Fc portion of human IgG and is available from Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA. Antibodies can be radiated iodinated using standard chromamine-T methods.

The antibody will bind to an elk / Fc or HEK / Fc fusion gene that binds to the cell. In all tests, the nonspecific binding of ¹²⁵ I antibody is tested in the presence and presence of elk / Fc (or HEK / Fc), 200-fold molar excess of anti-human IgG antibody in unlabeled mice.

The cell-bound ¹²⁵ I-antibody was quantitated on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. NY: Acad. Sci., 51: 660, 1949) were obtained on RS / 1 (BBN software, Boston, USA) operating on a microbox computer.

Example 3: Monoclonal antibody binding to Lurk-8 < RTI ID = 0.0 >

This example shows a method for producing a monoclonal antibody that binds to Lurk-8. Suitable immune genes that can be used to generate such antibodies include purified Lurk-8 proteins or fragments of the immunogens such as fusion proteins containing the extracellular domain or Lurk-8 (e.g., soluble Lurk-8 / Fc fusion protein But is not limited thereto.

Purified Lurk-8 can be used to generate monoclonal antibodies that are immunoreactive with conventional techniques such as those described in U.S. Patent No. 4,411,993. Briefly, mice are immunized with a luc-8 immunogen, which is emulsified in the complete adjuvant of Prund, followed by intravenous or subcutaneous injection in an amount of 10-100 μg. Animals immunized 10 to 12 days later are additionally promoted to Luck-8 with an adjuvant of incomplete Prand. The mice are then routinely promoted by the immunization schedule from week to week. Serum samples are routinely obtained by retro-orbital bleeding or tail-end resection to test for Lurk-8 antibodies, by a dot-blot test, ELISA (enzyme-linked immunosolvent test) or inhibition of Heck or elk binding.

After finding the appropriate concentration of the used antibody, a final intravenous injection of Lurk-8 in saline is provided to the positive animals. After 3 to 4 days, the animals are sacrificed and spleen cells are obtained and the spleen cells are fused to murine myeloid cell lines such as NS1, preferably P3 x 63 Ag 8.653 (ATCC CRL 1580). Fusion releases hybridoma cells placed on multiple microtiter plates in HAT (hypoxanthine, aminopterin and thymidine) selective medium which inhibits cleavage of hybrids of non-fused cells, myeloma hybrids and spleen cells.

Hybridoma cells were obtained from Engvall et al. Immunochem. 8: 871, 1971) and by ELISA, which is responsive to purified Lurk-8 by applying the technique disclosed in U.S. Patent No. 4,703,004. A preferred screen technique is the antibody capture technique described in Beekmann et al., (J. Immunol. 144: 4212, 1990). Positive hybridoma cells can be injected intraperitoneally into BALB / c mice producing multiple ascites (ascites) containing a high concentration of anti-Lurk-8 monoclonal antibody. Hybridoma cells can also be grown in vitro in an extracorporeal flask or roller bottle by a variety of techniques. The monoclonal antibody obtained from murine ascites may be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Affinity chromatography based on binding of the antibody to protein A or protein G can also be used, as can affinity chromatography based on binding to Lurk-8.

<Sequence Table>

(1) General information:

(i) Applicants: Cherti, Douglas P.

(ii) Title of the invention:

(iii) SEQ ID NO: 3

(iv) Communication address:

(A) Recipients: Kathryn A, Anderson, and Immunex Co operation

(B) Distance: 51 University Stittle

(C) City: Seattle

(D) Note: Washington

(E) Country: United States

(F) Postal Code: 98101

(v) Computer readable form:

(A) Media type: floppy diskette

(B) Computer: Apple Power Mae Kenthi

(C) Operating system: PC-DOS / MS-DOS

(D) Software: Apple Operating System 7.5.3

(vi) Recent application records:

(A) Application number: - Assigning -

(B) Filing Date: March 19, 1997

(C) Classification:

(viii) Agent information:

(A) Name: Anderson, Katrin Ai.

(B) Registration number: 32,172

(C) Reference number: 2839-WO

(ix) Communication information:

(A) Tel: (206) 587-0430

(B) Fax: (206) 233-0644

(C) Telex: 756822

(2) Information on sequence number 1:

(i) Sequence characteristics:

(A) Length: 1708 base pairs

(B) Type: Nucleic acid

(C) Number of strands: Single

(D) Topology: Linear

(ii) Molecular form: cDNA

(iii) Hypothesis: None

(iv) Antisense: None

(vii) Direct basis:

(B) Clone: Furuck 8

(ix) Features:

(A) Name / Key: CDS

(B) Location: 398..1420

(ix) Features:

(A) Name / Key: Methyl-peptide

(B) Location: 479..1417

(ix) Features:

(A) name / key: sig_peptide

(B) Location: 398..478

(ix) Sequence description: Sequence ID No. 1:

(2) Information on sequence number 2:

(i) Sequence characteristics:

(A) Length: 341 Amino acids

(B) Type: Amino acid

(C) Topology: Linear

(ii) Molecular form: protein

(xi) Sequence description: Sequence ID No. 2:

(2) Information on sequence number 3:

(i) Sequence characteristics:

(A) Length: 8 amino acids

(B) Type: Amino acid

(C) Number of strands: Single

(D) Topology: Linear

(ii) Molecular form: Peptide

(iii) Hypothesis: None

(iv) Antisense: None

(vii) Direct basis:

(B) Clone: FLAG peptide

(xi) Sequence description: Sequence ID No. 3:

Asp Tyr Lys Asp Asp Asp Asp Lys

Claims (27)

8 polypeptide comprising 80% or more identical amino acid sequence to a sequence selected from the group consisting of residues -27 to 313 of SEQ ID NO: 2 and residues 1 to 313 of SEQ ID NO: Isolated DNA that encodes. 2. The polypeptide of claim 1, wherein said Lurk-8 polypeptide comprises an amino acid sequence that is 90% or more identical to a sequence selected from the group consisting of residues -27 to 313 of SEQ ID NO: 2 and residues 1 to 313 of SEQ ID NO: DNA that is characterized by. 3. The DNA of claim 2, wherein said Lurk-8 polypeptide comprises an amino acid sequence selected from the group consisting of residues -27 to 313 of SEQ ID NO: 2 and residues 1 to 313 of SEQ ID NO: 3. The DNA of claim 1 or 2, wherein said Lurk-8 polypeptide occurs spontaneously. 1) the calculated molecular weight is about 33 kilodaltons, 2) the isoelectric point (pI) is about 8.46, 3) the N-terminal amino acid sequence is Leu-Ser-Leu-Glu-Pro-Val-Tyr-Trp-Asn- Ser- Ala- Asn- (amino acid 1-12 of Sequence Identification No. 2) 8 polypeptide that encodes a mature human lurk-8 polypeptide that binds to a HEK or an ELK, Heck or elk and comprises a sequence selected from the group consisting of residues -27 to x of SEQ ID NO: 2 (where x is an integer from 142 to 197) and residues 1 to x of SEQ ID NO: &Lt; RTI ID = 0.0 &gt;% &lt; / RTI &gt; identical amino acid sequence. 7. The isolated luciferase according to claim 6, wherein said soluble Lurk-8 polypeptide comprises an amino acid sequence selected from the group consisting of residues -27 to x of SEQ ID NO: 2 and residues 1 to x of SEQ ID NO: DNA, wherein x represents an integer of 142-197. 1) the human lurk-8 polypeptide of SEQ ID NO: 2; and 2) a polypeptide fragment of 1) capable of binding to an elk or a heck Lt; RTI ID = 0.0 &gt; luc-8 &lt; / RTI &gt; 9. The DNA of claim 8, wherein the fragment is a soluble fragment. 9. An expression vector comprising the DNA of any one of claims 1 to 9. 11. A host cell transformed with the expression vector of claim 10. 8. A method of producing a Lurk-8 polypeptide, comprising culturing the host cell of claim 11 under conditions that promote expression of the Lurk-8 polypeptide, and recovering the Lurk-8 polypeptide. 9. A purified Lurk-8 polypeptide encoded by the DNA of any one of claims 1 to 9. 8 polypeptides comprising an amino acid sequence that is 80% or more identical to the sequence of residues 1 to 313 of SEQ ID NO: 2. 15. A Lurk-8 polypeptide according to claim 14 comprising at least 90% identical amino acid sequence to the sequence of residues 1 to 313 of SEQ ID NO: 16. The Lurk-8 polypeptide of claim 15, comprising the amino acid sequence of residues 1 to 313 of SEQ ID NO: 16. The polypeptide of claim 15, wherein the residue at position 298 is a leucine Lurk-8 polypeptide comprising amino acids 1 to 297 and 299 to 313 of SEQ ID NO: Heck or elk, mature form 1) the calculated molecular weight is about 33 kilodaltons, 2) the isoelectric point (pI) is about 8.46, 3) the N-terminal amino acid sequence is Leu-Ser-Leu-Glu-Pro-Val-Tyr-Trp-Asn- Ser-Ala- Asn- (amino acid 1-12 of Sequence Identification No. 2) Purified human Lurk-8 protein. Heck or elk and comprises an amino acid sequence that is at least 80% identical to a sequence of residues 1 through x of SEQ ID NO: 2, wherein x represents an integer from 142 to 197. 20. The soluble luc-8 polypeptide of claim 19 comprising residues 1 through x of SEQ ID NO: 2, wherein x represents an integer from 142 to 197. 1) the human lurk-8 polypeptide of SEQ ID NO: 2; and 2) a polypeptide fragment of 1) capable of binding to an elk or a heck Lt; RTI ID = 0.0 &gt; luc-8 &lt; / RTI &gt; 22. The Lurk-8 polypeptide of claim 21, wherein said fragment is a soluble fragment. 22. An oligomer comprising 2 to 4 Lurk-8 polypeptides of any one of claims 13 to 22. 24. The oligomer of claim 23, wherein the oligomer is comprised of two soluble Lurk-8 / Fc fusion proteins. 24. A pharmaceutical composition comprising a Lact-8 polypeptide or oligomer of any one of claims 13 to 24, and a suitable diluent, excipient or carrier. 22. An antibody that acts on a Lurk-8 polypeptide of any one of claims 13 to 22. 27. The antibody of claim 26, wherein the antibody is a monoclonal antibody.