MXPA97009090A - Beta-11 human chemistry and chemisine alpha-1 hum - Google Patents

Abstract

The polypeptides of human chemokine and DNA (RNA) which codes for such chemokine polypeptides, and a method for the production of such polypeptides by recombinant techniques are described. Also described are methods for the use of such chemokine polypeptides for the treatment of leukemia, tumors, chronic infections, autoimmune diseases, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemokine polypeptides and their use as a therapeutic agent for treating rheumatoid arthritis, autoimmune diseases and chronic and acute inflammatory infections, allergic reactions, prostaglandin-independent fever and medullary failure are also described. Diagnostic assays for detecting diseases are also described in relation to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Diagnostic assays for detecting mutations in the polynucleotides encoding the chemokine polypeptides, and for detecting the levels of the polypeptide in a host are also described.

Description

CHEMISTRY BETA-11 HUMAN AND CHEMOTHERINE ALPHA-1 HUMAN DESCRIPTION OF THE INVENTION This invention relates to the newly identified polynucleotides, to the polynucleotides encoded by such polynucleotides, to the use of such polynucleotides and polypeptides, as well as to the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human chemokine polypeptides, sometimes referred to hereinbefore as human beta-11 chemokine (Ckß-11) and human alpha-1 chemokine (C a-1). The invention also relates to the inhibition of the action of such polypeptides. Chemokines, also called intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are of a size of 8-10 kd. In general, chemokines exhibit 20% to 75% homology at the amino acid level, and are characterized by four conserved cysteine residues that form two disulfide bridges. Based on the arrangement of the first two residues of REF: 26077 cysteine, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by an amino acid and hence are called co or the subfamilies "C-X-C". In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, termed the "C-C" subfamilies. By far, at least eight different members of this family have been identified in humans. The intercrine cytokines show a wide variety of functions. A distinctive feature is its ability to trigger the chemotactic migration of different cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adhesion of target or target immune cells to endothelial cells, increased linkage of complement proteins, induced expression of adhesion molecules of granulocytes and complement receptors, and respiratory burst. In addition to its involvement in inflammation, it has been shown that certain chemokines exhibit other activities. For example, the inflammatory protein 1 of macrophages (MIP-1) is capable of suppressing the proliferation of the cells of the hematopoietic line; platelet factor 4 (PF-4) is a potent inhibitor of the development of endothelial cells; Interleukin 3 (IL-8) promotes the proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells. In light of the various biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including the trafficking of lymphocytes, wound healing, hematopoietic regulation and immune disorders such as allergy. , asthma and arthritis. The members of the "C-C" branch exert their effects on the following cells: eosinophils, which destroy parasites to decrease parasitic infection, and cause chronic inflammation in the respiratory tract of the respiratory system; macrophages that suppress tumor formation in vertebrates; and the basophils which release histamine which plays a role in allergic inflammation. However, the members of one branch can exert an effect on the cells that normally respond to the other branch of the chemokines and, therefore, can not be assigned a precise role for the members of the branches. While the members of the C-C branches act predominantly on the mononuclear cells, and the members of the C-X-C branches act predominantly on the neutrophils, a chemoattractant property other than a chemokine can not be assigned based on this guideline. Some chemokines from one family show characteristics of the other. The polypeptides of the present invention have the conserved cysteine residues, namely Ckß-11 has CC regions, and Cka-1 has "CXC" regions, and these have high amino acid sequence homology to the known chemokines and therefore they have been optionally characterized as human chemokines. In accordance with one aspect of the present invention, novel polypeptides are provided which are Ckß-11 and Cka-1, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. According to yet another aspect of the present invention, the isolated nucleic acid molecules encoding such polypeptides are provided, including mRNAs, DNAs, cDNAs, genomic DNA, as well as diagnostically and therapeutically useful biologically active fragments, analogs and derivatives thereof. According to another aspect of the present invention, nucleic acid probes comprising nucleic acid molecules of sufficient length are provided to hybridize specifically to the sequences of Ckß-11 and Cka-11. According to yet another aspect of the present invention there is provided a process for the production of such polypeptides by recombinant techniques, which comprises culturing the prokaryotic and / or eukaryotic recombinant host cells comprising a nucleic acid sequence of Ckß-11 and Cka-1, under conditions that promote the expression of said protein and the subsequent recovery of the protein. According to yet another aspect of the present invention, there is provided a process for using such polypeptides, or polynucleotides encoding such polypeptides, for therapeutic purposes, for example, for treating solid tumors, chronic infections, leukemia, mediated autoimmune diseases. by T cells, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate the activity of growth factor, to inhibit angiogenesis and to promote wound healing. According to yet another aspect of the present invention, antibodies against such polypeptides are provided. According to yet another aspect of the present invention, antagonists are provided for such polypeptides, which can be used to inhibit the action of such polypeptides, for example, in the treatment of certain autoimmune diseases, atherosclerosis, inflammatory diseases and chronic infections. , allergic reactions mediated by histamine and IgG, prostaglandin-independent fever, disorders of the bone marrow, cancers, silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eosinophilic syndrome and fibrosis in the asthmatic lung.

According to yet another aspect of the present invention, there is provided a method for diagnosing a disease or a susceptibility to a disease related to a mutation in the nucleic acid sequences of Ckß-11 or Cka-1 and the protein encoded by such nucleic acid sequences. Accordingly - with yet another aspect of the present invention, there is provided a process for using such polypeptides, or the polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis and manufacturing DNA vectors. These and other aspects of the present invention should be apparent to those of skill in the art from the teachings contained herein. The following drawings are illustrative of the embodiments of the invention, and does not mean that they limit the scope of the invention, as encompassed by the claims. Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of Ckβ-11. The initial 17 amino acids represent the leader sequence, such that the putative mature polypeptide comprises 81 amino acids. Standard one-letter abbreviations for amino acids are used. Sequencing was performed using an automated DNA sequencer 373 (Applied Biosystems, Inc.). The sequencing accuracy is predicted to be greater than 97%. Figure 2 shows a cDNA sequence and the corresponding deduced amino acid sequence of Cka-1. The initial 22 amino acids represent the leader sequence, such that the putative mature polypeptide comprises 87 amino acids. Standard one-letter abbreviations for amino acids are used. According to one aspect of the present invention, there are provided the isolated nucleic acids (polynucleotides) which code for the mature polypeptides having the deduced amino acid sequences of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No 4) or for the mature polypeptides encoded by the cDNAs of the clones deposited as ATCC deposit No. 75948 (Ckß-11) and 75947 (Cka-1) on November 11, 1994. The polynucleotides encoding Ckß-11 can be isolated from numerous human and fetal cDNA genomic libraries, for example, a human fetal spleen cDNA library. Ckß-11 is a member of the C-C branch of chemokines. It contains an open reading frame which codes for a protein of 98 amino acid residues of which approximately the first 17 amino acid residues are the putative leader or leader sequence, such that the mature protein comprises 81 amino acids. The protein shows the highest degree of homology to the rat RANTES polypeptide with 31% identity and 47% similarity over a portion of 89 amino acids. It is also important that the four cysteine residues spatially conserved in the chemokines are found in the polypeptides. The polynucleotides that code for the Cka-1 can be isolated from the numerous libraries of fetal cDNA and human adult, for example, the cDNA library of human tonsils or tonsils. Cka-1 is a member of the C-X-C branch of chemokines. It contains an open reading frame which codes for a protein of 109 amino acid residues, of which approximately the first 22 amino acid residues are the putative leader or leader sequence, such that the mature protein comprises 87 amino acids. The protein shows the highest degree of homology to sheep interleukin 8 (Ovi e ari es) with 31% identity and 80% similarity over a portion of 97 amino acids. It is also important that the four cysteine residues spatially conserved in the chemokines be found in the polypeptides. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded, and if it is single-stranded, it can be the coding thread or the non-coding (antisense) thread. The coding sequence coding for the mature polypeptides may be identical to the coding sequences shown in Figures 1 (SEQ ID No. 1) and 2 (SEQ ID No. 3) or those of the deposited clones, or may be a different coding sequence, whose coding sequence, as a result of the redundancy or degeneracy of the genetic code, codes for the same mature polypeptides as the DNA of Figure 1 (SEQ ID No. 1) and 2 (SEQ ID No. 3) or the deposited cDNAs. Polynucleotides encoding the mature polypeptides of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the polypeptide mature; the coding sequence for the mature polypeptide and the additional coding sequence such as a guiding or secreting sequence, or a proprotein sequence; the coding sequence for * the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as the introns or the 5 'and / or 3' non-coding sequence of the coding sequence for the polypeptides mature. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only the coding sequence for the polypeptide, as well as a polynucleotide that includes the additional coding and / or non-coding sequence. The present invention further relates to the variants of the polynucleotides described hereinabove, which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequences of FIGS. 1 (SEQ ID No. 2) and FIG. (SEQ ID No. 4) or the encoded polypeptides of the cDNAs of the deposited clones. The variant of the polynucleotides can be a naturally occurring allelic variant of the polynucleotides, or a variant of non-natural origin of the polynucleotides. Thus, the present invention includes the polynucleotides encoding the same mature polypeptides * as those shown in Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or the same encoded mature polypeptides by the cDNA of the deposited clones, as well as the variants of such polypeptides, whose variants code for a fragment, derivative or analog of the polypeptides of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or the polypeptides encoded by the cDNA of the deposited clones. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As indicated hereinabove, the polynucleotides may have a coding sequence that is a naturally occurring allelic variant of the coding sequences shown in Figures 1 (SEQ ID No. 1) and 2 (SEQ ID No. 3) or of the coding sequence of the deposited clones. As is known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide. The present invention also includes polynucleotides, wherein the coding sequence for mature polypeptides can be fused in the same reading structure to a polynucleotide sequence that aids the expression and secretion of a polypeptide from a host cell, e.g. , a leader sequence that functions as a secretory sequence to control the transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein, and may have the leader sequence cleaved by the host cell, to form the mature form of the polypeptide. The polynucleotides may also code for a proprotein which is the mature protein, plus additional amino acid residues in the 5 'direction. A mature protein that has a prosequence is a proprotein and is in an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains.

Thus, for example, the polynucleotides of the present invention may code for a mature protein, or for a protein having a prosequence, or for a protein having a prosequence and a presequence (guide sequence). The polynucleotides of the present invention may also have the coding sequence fused in the structure to a marker sequence that allows purification of the polypeptides of the present invention. The marker sequence may be a histidine tag supplied by a PQE-9 vector, to provide for the purification of the mature polypeptides fused to the tag, in the case of a bacterial host, or for example, the tag sequence may be a hemagglutinin tag (HA) when a mammalian host is used, for example COS-7 cells. The HA tag responds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37: 767 (1984)). The term "gene" means the segment of DNA involved in the production of a polypeptide chain; it includes the preceding and following regions to the coding region (guide and tail) as well as the intervening sequence (introns) between the individual coding segments (exons). Fragments of the full-length gene of the present invention can be used as a hydride probe for a cDNA library, to isolate the full length cDNA and to isolate other cDNAs having a high sequence similarity to the gene, or similar activity biological Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe can also be used to identify a cDNA clone corresponding to a full-length transcript and a clone or genomic clones containing the complete gene, including, the regulatory and promoter regions, the exons and the introns. An example of the selection comprises the isolation of the coding region of the gene, by using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a genomic library of human cDNA, genomic DNA or mRNA, to determine which members of the genomic library the probe hybridizes to.

The present invention further relates to polynucleotides that hybridize to the sequences described hereinabove, if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention relates particularly to polynucleotides that hybridize under stringent conditions to the polynucleotides described above. As used herein, the term "stringent conditions" means that hybridization will occur only if there is at least 95%, and preferably at least 97% identity between the sequences. Polynucleotides that hybridize to the polynucleotides described hereinbefore, in a preferred embodiment, encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID No. 1) or the deposited cDNAs. Alternatively, the polynucleotide can have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 bases that hybridize to a polynucleotide of the present invention, and have an identity thereto, as described hereinabove. , and which may or may not retain the activity. For example, such polynucleotides can be used as probes for the polynucleotide of SEQ ID No. 1, for example, for the recovery of the polynucleotides or as a diagnostic probe or as a PCR primer. Thus, the present invention is directed to polynucleotides having at least 70% identity, preferably at least 90% and more preferably at least 95% identity to a polynucleotide encoding the polypeptide of SEQ ID No. 2, as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases , and the polypeptides encoded by such polynucleotides. The deposit (s) referred to herein will be maintained under the terms of the Budapest treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit under 35 U.S.C. Section 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequences of the polypeptides encoded by them, are incorporated herein by reference and are control in the case of any conflict with any description of sequences in the present. A license to manufacture, use or sell the deposited materials may be required, and no such license is hereby granted. The present invention further relates to polypeptides having the deduced amino acid sequences of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or which have the amino acid sequence encoded by the deposited cDNA , as well as fragments, analogues and derivatives of such polypeptides. The terms "fragment", "derivative" and "analogue" when referring to the polypeptides of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or that encoded by the deposited cDNA, mean polypeptides that they retain essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein that can be activated by cleavage of the proprotein portion, to produce an active mature polypeptide. The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides. The fragment, derivative or analog of the polypeptides of Figures 1 (SEQ ID No. 2) and 2 (SEQ ID No. 4) or that encoded by the deposited cDNAs can be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue * (preferably a conserved amino acid residue) and such a substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more than the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused to another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a guiding or secretory sequence, or a sequence that is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives or analogs are considered to be within the scope of those skilled in the art from the teachings described herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are preferably purified to homogeneity. The term "isolated" means that the material is removed from its original environment (for example, the natural environment if it is of natural origin). For example, a polynucleotide or polypeptide of natural origin present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector, and / or such polynucleotides or polypeptides could be part of a composition, and still be isolated even if such vector or composition is not part of their natural environment. The polypeptides of the present invention include the polypeptides of SEQ ID No. 2 (in particular the mature polypeptide) as the polypeptides having at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID No 2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID No. 2, and still more preferably at least 95% similarity (still more preferably at least 95% of identity) to the polypeptide of SEQ ID No. 2, and also include portions of such polypeptides with such a portion of the polypeptide that generally contains at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequences and their conserved amino acid substitutes of a polypeptide to the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention can be employed to produce the corresponding full-length polypeptide by peptide synthesis; Fragments can be used as intermediates in the production of full-length polypeptides. Fragments or portions of the polynucleotides of the present invention can be used to synthesize the full length polynucleotides of the present invention. The present invention also relates to vectors that include polynucleotides of the present invention, host cells that are engineered with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. The host cells are engineered (transduced or transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. Genetically engineered host cells can be cultured in conventional nutrient media, modified as appropriate for promoter activation, transformant selection or amplification of the Ckß-11 or Cka-1 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those of ordinary skill in the art. The polynucleotides of the present invention can be used for the production of polypeptides by recombinant techniques. Thus, for example, the polynucleotide can be included in any of a variety of expression vectors for the expression of a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; Phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia virus, adenovirus, poultry virus of domestic poultry, and pseudorabies. However, any other vector can be used as long as it is replicable and viable in the host. The appropriate sequence of DNA can be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into one or several appropriate restriction endonuclease sites by methods known in the art. Such procedures and others are considered to be within the scope of those of skill in the art. The DNA sequence in the expression vector is operably linked to one or more expression control sequences, appropriate, promoters, to direct the synthesis of mRNA. As a representative example of such promoters, there may be mentioned: the LTR or SV40 promoters, lac or trp of E. coli, the PL promoter of phage lambda and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells, or their virus. The expression vector also contains a ribosome binding site for the initiation of translation, and a transcription terminator. The vector may also include appropriate sequences for the amplification of expression. In addition, the expression vectors preferably contain one or more selectable marker genes, to provide a phenotypic trait for the selection of transformed host cells such as resistance to dihydrofolate reductase or neomycin for the culture of eukaryotic cells, or such such as resistance to tetracycline or ampicillin in E. coli. The vector containing the appropriate DNA sequence as described hereinabove, as well as an appropriate promoter or appropriate control sequence, can be employed to transform an appropriate host, to allow the host to express the protein. As representative examples of the appropriate hosts, there may be mentioned: bacterial cells, such as E coli, Streptomyces, Salmonella typhimuri um; mushroom cells, such as yeast; insect cells such as Drosophil to S2 and Spodopt was Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. The selection of an appropriate host is considered within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as are widely described herein. The constructs comprise a vector, such as a plasmid or viral vector, within which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises the regulatory sequences, including, for example, a promoter operably linked to the sequence. A large number of appropriate vectors and appropriate promoters are known to those skilled in the art, and are commercially available. The following vectors are provided by way of example. Bacterials: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pBleuescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used, as long as they are replicable and viable in the host. The promoter regions can be selected from any desired gene using the CAT vectors (chloroanfenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. The named, particular bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda Pf, PL and trp. Eukaryotic promoters include early immediate CMV, HSV thi idine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-I. The selection of the appropriate promoter and vector is within the level of ordinary skill in the art. In a further embodiment, the present invention relates to host cells containing the constructions described above. The host cells can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology , (1986)). Constructs in the host cells can be used in a conventional manner to produce the genetic products encoded by the recombinant sequences. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Cold Spring Harbor, N. Y., (1989), the disclosure of which is incorporated by reference herein. The transcription of the DNA coding for the polypeptides of the present invention, by higher eukaryotes, is increased by the insertion of an autative sequence within the vector. Authors are elements that act in cis position of DNA, usually from approximately 10 to 300 base pairs that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin of 100 to 270 base pairs, an early or early cytomegalovirus promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers . In general, recombinant expression vectors will include origins of replication and selectable markers that allow the transformation of the host cell, for example, the ampicillin resistance gene of the TRP1 gene of E. coli and S. cerevi siae, and a promoter derived from a gene highly expressed to direct the transcription of a 3 'structural sequence. Such promoters can be derived from operons that code for glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), factor a, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in the appropriate phase with the translation initiation or termination sequences, and preferably, a guiding sequence capable of directing the secretion of the produced protein towards the periplasmic space or towards the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that imparts the desired characteristics, for example, stabilization or simplified purification of the expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein, together with the appropriate start and end signals of production, in operable reading phase with a functional promoter. . The vector will comprise one or more phenotypic selectable markers and an origin of replication, to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacill? s subtili s, Salmonella typhimuri um and various species within the genera Pseudomonas, Streptomyces, and Staphi l ococcus, although others may also be employed as a materi * a of choice. As a representative but not limiting example, expression vectors useful for bacterial use may comprise a selectable marker and the bacterial origin of replication derived from commercially available plasmids, which comprise genetic elements of the cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, Wl, USA). These "backbone" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of an appropriate host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature change or chemical induction) and the cells are cultured for an additional period. . The cells are typically harvested by centrifugation, broken by physical or chemical means, and the resulting crude extract is retained for further purification. The microbial cells employed in the expression of proteins can be destroyed by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysis agents, such methods being well known to those skilled in the art. Various mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 monkey kidney fibroblast lines described by Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing a compatible vector, e.g., cell lines C127, 3T3, CHO, HeLa and BHK. Mammalian expression vectors will comprise an origin of replication, an appropriate promoter and enhancer, and also any necessary chromosome binding sites, a polyadenylation site, donor and splice acceptor sites, transcriptional termination sequences and sequences not transcribed at the 5 'end. The DNA sequences derived from the SV40 splice, and the polyadenylation sites, can be used to provide the required non-transcribed genetic elements. Polypeptides can be recovered and purified from recombinant cell cultures by methods that include precipitation with ammonium sulfate or ethanol, acid extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, chromatography with hydroxyapatite and chromatography with lectin. Protein refolding steps may be used, as necessary, to complete the mature protein configuration. Finally, high performance liquid chromatography (HPLC) can be used for the final purification steps. The polypeptides of the present invention may be a naturally purified product or a product of synthetic chemical processes, or produced by recombinant techniques from a prokaryotic or eukaryotic host (e.g., by bacterial, yeast, higher plant, insect cells). and of mammal in crops). Depending on the host employed in a recombinant production method, the polypeptides of the present invention can be glycosylated, or they can be non-glycosylated. The polypeptides of the invention may also include * an amino acid residue with initial methionine. The polynucleotides and polypeptides of the present invention can be used as research reagents and materials for the discovery of treatments and diagnostics for human diseases. Human chemokine polypeptides can be used to inhibit the formation of totipotent cell colonies of the bone marrow, as adjunctive protective treatment during cancer chemotherapy, and for leukemia. Polypeptides of human chemokine can also be employed to inhibit the proliferation of epidermal keratinocytes for the treatment of psoriasis, which is characterized by hyperproliferation of keratinocytes.

Human chemokine polypeptides can also be used to treat solid tumors by stimulating the invasion and activation of host defense cells, cytotoxic T cells and macrophages, and by the inhibition of tumor angiogenesis. These can also be used to improve host defenses against chronic infections * and acute resistant ones, for example, mycobacterial infections through the attraction and activation of microbicidal leukocytes. Human chemokine polypeptides can also be used to inhibit T cell proliferation, by inhibiting the biosynthesis of interleukin 2 for the treatment of autoimmune diseases mediated by T cells, and lymphocytic leukemias. Ckß-11 and Cka-1 can also be used to stimulate wound healing, either via the recruitment of inflammatory cells that promote the clearance of debris and connective tissue, and also via their control of TGFβ-mediated fibrosis. excessive In the same way, Ckß-11 and Cka-11 can also be used to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis.

Human chemokine polypeptides also increase the presence of eosinophils, which have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis. These can also be used to regulate hematopoiesis, by regulating the activation and differentiation of various hemotopoietic progenitor cells, for example, to release mature leukocytes from the bone marrow after chemotherapy. The polynucleotides and the polypeptides encoded by such polynucleotides can also be used for in vi tro purposes related to scientific research, for the synthesis of DNA and manufacture of DNA vectors, and for the design of therapeutics and diagnostics for the treatment of human diseases. . Fragments of full-length Ckß-11 or Cka-1 genes can also be used, with a hybridization probe for a cDNA library, to isolate the full-length gene, and to isolate other genes that have high sequential similarity to the gene or similar biological activity. Probes of this type generally have at least 20 bases.

Preferably, however, the probes have at least 30 bases and generally do not exceed 50 bases, although these may have a higher number of bases. The probe can also be used to identify a cDNA clone corresponding to a full-length transcript, and a clone or genomic clones containing the complete genes, including the regulatory and promoter regions, exons and introns. An example of a selection comprises isolating the coding region of the genes by using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to those of the genes of the present invention are used to select a genomic library of human cDNA, genomic DNA or mRNA, to determine which members of the library the probe hybridizes to. This invention is also related to the use of the Ckβ-11 and Cka-1 gene as part of a diagnostic assay to detect diseases or the susceptibility of diseases related to the presence of mutations in the nucleic acid sequences of Ckß-11 or Cka -1. Such diseases are related to the under-expression of human chemokine polypeptides, for example, tumors and cancers.

Individuals carrying mutations in the Ckß-11 or Cka-1 gene can be detected at the DNA level by a variety of techniques. The nucleic acids for diagnosis can be obtained from the cells of a patient, such as blood material, urine, saliva, tissue biopsy and autopsy. Genomic DNA can be used directly for detection or can be amplified enzymatically by using PCR (Sakai et al., Nature, 324: 163-166 (1986)) before analysis. The cDNA or RNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding Ckß-11 or Cka-1 can be used to identify and analyze mutations of Ckß-11 or Cka-1. For example, deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridization of amplified DNA to radiolabeled Ckβ-11 or Cka-1 RNA or alternatively, radiolabeled Ckβ-11 or Cka-1 anti-sense DNA sequences. The perfectly coupled sequences can be distinguished from uncoupled duplexes by digestion with RNase A or by differences in melting temperatures. The genetic test based on the differences of the DNA sequences can be carried out by detecting the alteration in the electrophoretic mobility of DNA in genes with or without denaturing agents. Small deletions or sequential insertions can be visualized by high resolution gel electrophoresis. The DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels, in which the mobilities of the different DNA fragments are recharged in the gel at different positions according to their specific melting or partial melting temperatures (see , for example Myers et al., Science, 230: 1242 (1985)). Sequential changes at specific sites can also be revealed by nuclease protection assays, such as protection with RNAse and SI, or the chemical cleavage method (eg, Cotton et al., PNAS, USA, 85: 4397-4401 ( 1985)). Thus, the detection of a specific DNA sequence can be achieved by methods such as hybridization, RNAse protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg, Restriction Fragment Length Polymorphisms). , (RFLP)) and spotting or Southern blotting of genomic DNA. In addition to more conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in si t u analysis. The present invention also relates to a diagnostic assay or assay for detecting altered levels of the Ckß-11 or Cka-1 protein in various tissues, since an overexpression of the proteins compared to normal control tissue samples can detect the presence of a disease or susceptibility to a disease, for example, a tumor. The assays used to detect levels of the Ckß-11 or Cka-1 protein in a sample derived from a host are well known to those of skill in the art, and include radioimmunoassays, competitive binding assay, transfer analysis of Western, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Imology, 1 (2), Chapter 6, (1991)) initially comprises the preparation of an antibody specific for the Ckß-11 or Cka-1 antigen, preferably a monoclonal antibody. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme * is attached to the reporter antibody. * A sample of a host is removed and incubated on a solid support, for example, a polystyrene disk that binds to the proteins in the sample. Any protein binding sites, free on the disk, are then covered by incubation with a non-specific protein such as BSA. Next, the monoclonal antibody is incubated in the disk, during which time the monoclonal antibodies are coupled to any of the Ckß-11 or Cka-1 proteins coupled to the polystyrene disk. All unbound monoclonal antibody is washed with buffer. The reporter antibody linked to horseradish peroxidase is now placed on the disk, which results in the binding of the reporter antibody to any monoclonal antibody bound to Ckß-11 or Cka-1. The uncoupled reporter antibody is washed. The peroxidase substrates are then added to the disk and the amount of the color developed in a given period of time is a measure of the amount of Ckß-11 or Cka-1 proteins present in a given volume of the patient sample, when compare against a standard curve. A competition assay may be employed, where antibodies specific to Ckß-11 or Cka-1 are coupled to a solid support and labeled Ckß-11 or Cka-1, and a sample derived from Western is passed on a solid support , and the detected amount of marker, for example, by liquid scintillation chromatography, can be correlated to an amount of Ckβ-11 or Cka-1 in the sample. A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay Ckß-11 or Cka-1 is passed over a solid support and binds to the antibody bound to a solid support. A second antibody is then linked to Ckß-11 or Cka-1. A third antibody that is labeled and specific to the second antibody is then passed over the solid support and bound to the second antibody, and an amount can then be quantified. This invention provides a method for the identification of receptors for human chemokine polypeptides. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example panning of the ligand and FACS sorting (Coligan, et al., Current Protocole in Immun., 1 (2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to the polypeptides, and a cDNA genomic library created from this RNA is divided into pools and used to transfect COS or other cells. cells that do not respond to polypeptides. Transfected cells that develop on glass slides are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subject to autoradiographic analysis. The positive combinations are identified, and the sub-combinations are prepared and transfected using an iterative sub-combination and re-selection process, eventually producing a simple clone that encodes the putative receptor.

As an alternative method for identification of the receptor, the labeled polypeptides can be ligated by photoaffinity with the cell membrane or with extract preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subject to protein microsequencing. microsequencing could be used to designate a group of degenerate oligonucleotide probes to select a cDNA genomic library to identify the genes encoding the putative receptors This invention provides a method of selecting the compounds to identify agonists and antagonists for the polypeptides of human chemokine of the present invention An agonist is a compound that has similar biological functions of the polypeptides, whereas antagonists block such functions Chemotaxis can be assayed or assessed by placing the cells, which are chemoattracted by any of the polypeptides of the present invention, on top of a filter with pores of sufficient diameter to admit cells (approximately 5 μm). The solutions of the potential agonists are placed at the bottom of the chamber with an appropriate control means in the upper compartment, and in this way a concentration gradient of the agonist is measured, by counting the cells migrating to or through the porous membrane, over time. When evaluating the antagonists, the human chemokine polypeptides of the present invention are placed in the lower chamber and the potential antagonist is added to determine if the chemotaxis of the cells is prevented. Alternatively, a mammalian cell or a membrane preparation expressing the polypeptide receptors could be incubated with a human chemokine polypeptide, labeled, for example with radioactivity, in the presence of the compound. The ability of the compound to block this interaction could then be measured. When agonists are tested or evaluated in this way, human chemokines would be absent, and the ability of the agonist itself to interact with the receptor could be measured.

Examples of potential antagonists of Ckβ-11 and Cka-1 include antibodies, in some cases, oligonucleotides, which bind to the polypeptides. Another example of a potential antagonist is a dominant negative mutant of the polypeptides. Dominant negative mutants are polypeptides that bind to the wild-type polypeptide receptor, but can not retain biological activity. Anti-sense constructs prepared using anti-sense technology are also potential antagonists. The anti-sense technology can be used to control the expression of the gene through the formation of triple helix or DNA or anti-sense RNA, whose methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which codes for the mature polypeptides of the present invention, is used to designate an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix, see Lee et al., Nucí Acids, Res 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991)), thereby preventing the transcription and production of human chemokine polypeptides. The antisense RNA oligonucleotide hybridizes to the mRNA in vi and blocks the translation of the mRNA molecule into the polypeptides (anti-sense, Okano, J. Neurochem, 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be distributed to the cells such that the anti-sense RNA or DNA can be expressed in order to inhibit the expression of the human chemokine polypeptides. Another potential antagonist of human chemokine is a peptide derivative of polypeptides that are naturally or synthetically modified analogs of polypeptides that have lost biological function, which still recognize and bind to polypeptide receptors to effectively block this. the receivers. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules. The antagonists can be used to inhibit the chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some subgroups of T cells, for example, cytotoxic and activated CD8 T cells, and natural killer cells, in certain autoimmune, inflammatory and infectious chronic diseases. Examples of autoimmune diseases include multiple sclerosis, and insulin-dependent diabetes. Antagonists can also be used to treat infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis, by preventing and recruiting the activation of mononuclear phagocytes. These can also be used to treat idiopathic hypereosinophilic syndrome by preventing the production and migration of eosinophils. The endotoxic shock can also be treated by the antagonists, by preventing the migration of the macrophages and their production of the human chemokine polypeptides of the present invention. Antagonists can also be used to treat atherosclerosis, to prevent the infiltration of monocytes into the arterial wall. Antagonists can also be used to treat histamine-mediated allergic reactions and immune disorders including late-phase allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced degranulation of basophils and mast cells, and the release of histamine. . IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema can also be treated. Antagonists can also be used to treat chronic and acute inflammation, by preventing the attraction of monocytes to a wounded area. These can also be used to regulate normal populations of pulmonary macrophages, since chronic and acute inflammatory pulmonary diseases are associated with the sequestration of mononuclear phagocytes in the lung. They can also be used antagonists to treat rheumatoid arthritis, by preventing the attraction of monocytes to synovial fluid in the joints of patients. The influx and activation of monocytes plays a significant role in the pathogenesis of degenerative and inflammatory arthropathies. Antagonists can also be used to interfere with harmful cascades attributed mainly to IL-1 and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, antagonists can be used to prevent inflammation. Antagonists can also be used to inhibit prostaglandin-independent fever, induced by chemokines. The antagonists can also be used to treat cases of bone marrow deficiency, for example, aplastic anemia and myelodysplastic syndrome. Antagonists can also be used to treat asthma and allergy by preventing the accumulation of eosinophils in the lung. Antagonists can also be used to treat fibrosis of the subepithelial basement membrane, which is a prominent feature of the asthmatic lung. Antagonists can also be employed in a composition with a pharmaceutically acceptable carrier, for example, as described later herein. The human chemokine polypeptides and the agonists and antagonists can be used in combination with an appropriate pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be appropriate for the mode of administration. The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the containers of the pharmaceutical compositions of the invention. Associated with such containers may be a notice in the form prescribed by a governmental agency, which regulates the manufacture, use or sale of pharmaceutical or biological products, whose notice reflects the approval by the agency, the manufacture, the use or the sale for human administration. In addition, polypeptides and agonists and antagonists can be used in conjunction with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner such as by topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal, or intradermal routes. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In general, the polypeptides will be administered in an amount of at least 10 μg / kg of body weight, and in most cases will be administered in an amount not greater than about 8 mg / kg of body weight per day. In most cases, the dose is from about 10 μg / kg to about 1 mg / kg in body weight daily, taking into account the routes of administration, symptoms, etc. Human chemokine polypeptides and agonists or antagonists that are polypeptides can be employed according to the present invention by expressing such polypeptides in vi, which is often referred to as "gene therapy". Thus, for example, cells from a patient can be genetically engineered with a polynucleotide (DNA or RNA) that codes for an ex vivo polypeptide, with cells engineered that are then provided to a patient to be treated with the polypeptide. Such methods are well known in the art. For example, cells can be engineered by methods known in the art, by using a retroviral particle containing RNA encoding a polypeptide of the present invention. Similarly, cells can be engineered by in vitro gene for expression of an in vi ve polypeptide, by, for example, procedures known in the art. As is known in the art, a producer cell for the production of a retroviral particle containing RNA encoding the polypeptide of the present invention can be administered to a patient for genetic engineering of the cells in vi and the expression of the polypeptide in vi vo. This and other methods for the administration of a polypeptide of the present invention, by such method, should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for genetically engineering the cells may be different from a retrovirus, for example, an adenovirus that can be used to engineer the cells in vi, after combination with a delivery vehicle. appropriate.

Retroviruses from which the retroviral plasmid vectors mentioned hereinabove can be derived, include, but are not limited to, the Moloney murine leukemia virus, splenic necrosis virus, retroviruses such as sarcoma virus, Rous, Harvey's sarcoma virus, avian leukosis virus, gibbon monkey lupus eukaryotic virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus. The vector includes one or more promoters. Suitable promoters that can be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter, described in Miller et al., Biotechniques, vol. 7, N °. 9, 980-990 (1989), or any other promoter (for example, cellular promoters such as eukaryotic cell promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters that can be employed include, but are not limited to, adenoviral promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The selection of an appropriate promoter will be apparent to those skilled in the art from the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention is under the control of an appropriate promoter. Suitable promoters that can be employed include, but are not limited to, adenoviral promoters, such as the late adenoviral major promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters, the albumin promoter, the ApoAI promoter; the promoters of human globin; thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; Retroviral LTRs (including the modified retroviral LTRs described hereinabove); the β-actin promoter; and the promoters of human growth hormone. The promoter may also be the native promoter that controls the gene encoding the polypeptide.

The retroviral plasmid vector is used to transduce the packaging cell lines, to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, cell lines, PE501, PA317,? -2,? -AM, PA12, T19-14X, VT-19-17-H2,? CRE ,? CRIP, -GP + E-86, * GP-envAml2, and DAN, as described in Miller, Human Gene Therapy, vol. 1, pages. 5-14 (1990), which is incorporated by reference herein in its entirety. The vector can transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and calcium phosphate precipitation. In an alternative, the retroviral plasmid vector can be encapsulated within a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious particles of the retroviral vector, which include the nucleic acid sequence (s) encoding the polypeptides. Such retroviral vector particles can then be used to transduce the eukaryotic cells, either in vi tro or in vi vo. The transduced eukaryotic cells will express the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic totipotent cells, embryonic carcinoma cells, as well as totipotential hematopoietic cells., hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is specifically designated and can hybridize to a particular site on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosome. Currently very few chromosome labeling reagents are available, based on the effective sequence data (repetitive polymorphisms), for the chromosomal site labeling. The elaboration of the maps of the DNAs to the chromosomes according to the present invention is an important first step in the correlation of these sequences with the genes associated with the disease. Briefly, the sequences can be mapped to the chromosomes by preparing PCR primers (preferably 15-25 base pairs) from the cDNA. Computer analysis of the 3 'untranslated region is used to rapidly select primers that do not encompass more than one exon in the genomic DNA, thus complicating the amplification processes. These primers are then used for the selection by PCR of hybrids from somatic cells, which contain individual human chromosomes. Only those hybrids that contain the human gene that corresponds to the primer will produce an amplified fragment. The elaboration of the map by the PCR of the hybrids of somatic cells, is a fast procedure for the assignment of a particular DNA to a particular chromosome. Using the present invention with the oligonucleotide primers, sublocation can be accomplished with panels of fragments from specific or combined chromosomes of large genomic clones in an analogous manner. Other mapmaking strategies, which can be similarly used to map the chromosome, include in si t u hybridization, preselecting with labeled chromosomes, classified by flow, and pre-selection by hybridization to build chromosome-specific cDNA libraries. In si t u hybridization by fluorescence (FISH) from a cDNA clone to a metaphase chromosome spread can be used to provide a precise chromosome site in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Cromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to an accurate chromosome site, the physical position of the sequence on the chromosome can be correlated with the genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins Univesity Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through the analysis of (co-inheritance of physically adjacent genes). Next, it is necessary to determine the difference in the cDNA sequence or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any of the normal individuals, then the mutation is likely to be the causative agent of the disease. With the current resolution of the elaboration of the physical map and the techniques of the elaboration of the genetic map, a cDNA * located precisely for a chromosomal region associated with the disease, could be one of between 50 and 500 potential causal genes. (This assumes a map making resolution of one megabase and one gene per 20 kb). Polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them, can be used as an immunogen to produce antibodies for these. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be employed for the production of such antibodies and fragments.

The antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a non-human one. The antibody thus obtained will then bind to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies that bind to the entire native polypeptides. Such antibodies can then be used to isolate the polypeptide from the tissue expressing that polypeptide. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cultures of the cell line can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256: 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72). , and the EBV hybridoma technique to produce human monoclonal antibodies (Colé, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R., Liss, Inc. pp. 77-96). The techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce the single chain antibodies for immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express humanized antibodies to the immunogenic polypeptides of this invention. The present invention will be further described with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. In order to facilitate the understanding of the following examples, certain methods and / or terms of frequent occurrence will be described. "Plasmids" are designated by a p preceded and / or followed by capital letters and / or numbers. The initial plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to the person of ordinary skill in the art. "Digestion" of DNA refers to the cleavage or catalytic breakdown of DNA with a restriction enzyme that acts only on certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as it might be known to the person of ordinary skill in the art. For analytical reasons, typically 1 μg of plasmid or DNA fragment with approximately 2 units of enzyme is used in approximately 20 μl of buffer. For the purpose of isolating the DNA fragments for the construction of the plasmid, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. The appropriate buffers and the amounts of substrate for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C are ordinarily used, but may vary according to the supplier's instructions. After digestion the reaction is subjected to electrophoresis directly on a polyacrylamide gel, to isolate a desired fragment. The size separation of the excised fragment is performed using 8 percent polyacrylamide gel, described by Goeddel, D. et al., Nucleic Acids Res., 8: 4057 (1980). "Oligonucleotides" refers to either a single chain polydeoxynucleotide or two complementary polydeoxynucleotide strands, which can be chemically synthesized. Such synthetic oligonucleotides do not have phosphate in the 5 'position and thus will not be ligated to another oligonucleotide without the addition of phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligature" refers to the process of formation of phosphodiester bonds between two double-stranded nucleic acid fragments (Maniatis, T., et al., Id., Page 146). Unless otherwise provided, ligation can be performed using known buffers and conditions, with 10 units for T4 DNA ligase ("ligase") with only 0.5 μg of approximately equimolar amounts of the DNA fragments. to be linked. Unless stated otherwise, the transformation was performed as described in the method of Graham, F. and Van der Eb, A. Virology, 52: 456-457 (1973).

Example 1 Bacterial Expression and Purification of Ckß-11 The DNA sequence encoding Ckβ-11, ATCC # 75948, is initially amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' end sequences of the processed Ckß-11 nucleic acid sequence (minus the putative signal peptide sequence). Additional nucleotides corresponding to the Ckß-11 gene are added to the 5 'and 3' end sequences respectively. The 5 'oligonucleotide primer has the sequence 5'-CCCGCATGCCAACTCTGAGTGGCACCA-3', contains a Sphl restriction enzyme site (bold) followed by 18 nucleotides of the Ckß-11 coding sequence (underlined) starting from the second nucleotide of the sequences that code for the mature protein. The ATG codon is included in the Sphl site. In the next codon after the ATG, the first base is from the Sphl site and the two remaining bases correspond to the second and third bases of the first codon (residue 18) of the putative mature protein. The 3 '5'-CCCGGATCCCAATGCTTGACTCGGACT 3' sequence contains the sequences complementary to a BamH1 site (bold) and is followed by 18 nucleotides of the gene-specific sequences preceding the stop codon. The restriction enzyme sites corresponding to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 codes for the antibiotic resistance (Amp1) a bacterial origin of replication (ori), an IPTG (P / O) regulatable promoter operator, a ribosome binding site (RBS), a 6-His tag and the restriction enzyme sites. pQE-9 is then digested with Sphl and BamHl. The amplified sequences are ligated into pQE-9 and are inserted into the structure in the sequence encoding the histidine tag and the RBS. The ligation mixture is then used to transform the M15 / rep 4 strain of E. coli (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, ( 1989). M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance against kanamycin (Kanr). Transformants are used for their ability to grow on LB plates, and colonies resistant to ampicillin / kanamycin are selected. The plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (O / N) in liquid culture in LB medium supplemented with either Amp (110 μg / ml) and Kan (25 μg / ml). The O / N is used to inoculate a large crop at a ratio of 1: 100 to 1: 250. The cells are developed at an optical density 600 (O.D.fc0 °) between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto-pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivation of the lacl repressor, the clearance of P / 0 which leads to the increased expression of the gene. The cells develop an additional 3 to 4 hours. The cells are then harvested by centrifugation. The cell button is solubilized in the chaotropic agent 6 molar guanidine hydrochloride pH 5.0 After clarification, solubilized Ckβ-11 is purified from this solution by chromatography on a nickel chelate column under conditions that allow strong binding by proteins containing the 6-His tag (Hochuli, R. et al., J. Chromatography 411: 177-184 (1984)). Ckβ-11 (with purity> 98%) is eluted from the column in 6 M guanidine hydrochloride. Re-naturalization of the protein out of GnHCl can be carried out by various protocols (Jaenicke, R. and Rudolph, R., Protein Structure A Practical Approach, IRL Press, New York (1990)). Initially, the dialysis step is used to eliminate guanidine hydrochloride. Alternatively, the purified protein isolated from the Ni-chelate column can be joined to a second column on which a linear gradient in decrease of guanidine hydrochloride is run. The protein is allowed to renature while bound to the column, and is subsequently eluted with a buffer containing 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl, pH 7.5 and 10% glycerol. Finally, the soluble protein is dialyzed against a storage buffer containing 5 mM ammonium acid carbonate.

Example 2 Bacterial Expression and Purification of Cka-1 The DNA sequence encoding Cka-1, ATCC # 75947, is initially amplified using the oligonucleotide PCR primers corresponding to the sequences of the 5 'and 3' ends of the processed Cka-1 nucleic acid sequence (minus the putative signal peptide sequence). Additional nucleotides corresponding to Cka-1 are added to the 5 'and 3' end sequences respectively. The 5 'oligonucleotide primer having the sequence 5'-CCCGCATGCCTTCTGGAGGTCTATTACACA-3', contains a Sph1 restriction enzyme site (bold) followed by 21 nucleotides of the sequence coding for Cka-1 starting from the second nucleotide of the sequences that code for the mature protein. An ATG codon is included in the Sphl site. In the next codon after the ATG, the first base is the Sphl site and the remaining two bases correspond to the second and third bases of the first codon (residue 23) of the putative mature protein. As a consequence, the first base in this codon is changed from G to C as compared to the original sequences, resulting in a Val to Leu substitution in the recombinant protein. The sequence 3 ', 5' -CCCGGATCCGGGAATCTTTCTCTTAAAC-3 'contains sequences * complementary to the BamH1 site (bold) and is followed by the 19 nucleotides of the gene-specific sequences preceding the stop codon. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 encoding resistance to the antibiotic (Ampr), an origin of the bacterial application (ori), an IPTG (P / O) regulator promoter operator, a ribosome binding site (RBS), a 6-His tag and the restriction enzyme sites. pQE-9 is then digested with Sphl and BamHl. The amplified sequences are ligated into pQE-9 and inserted into the structure with the sequence encoding the histidine tag and the RBS. The ligation mixture is then used to transform E. coli M15 / rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance against kanamycin (Kan1 '). Transformants are identified by their ability to grow on LB plates and resistant columns * to * ampicillin / kanamycin are selected. The plasmid DNA is isolated and confirmed by restriction analysis. The clones containing the desired constructs are grown overnight (O / N) in liquid culture in LB medium supplemented with either Amp (100 μg / ml) and Kan (25 μg (ml).) The O / N culture is used. to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells are developed at an optical density at 600 (OD ') between 0.4 and 0.6 IPTG ("Isopropyl-BD-thiogalactopyranoside") is then added. at a final concentration of 1 mM IPTG induces the clearance of P / O by inactivation of the lacl repressor, which leads to increased expression of the gene.The cells are grown 3 to 4 additional hours.The cells are then harvested by centrifugation. The cellular button is solubilized in the chaotropic agent 6 molar guanidine hydrochloride, pH 5.0 After the clarification, solubilized Cka-1 is purified from this solution by chromatography on a nickel chelate column under conditions that allow strong binding or part of the proteins containing the 6-His tag (Hochuli, R. et al., J. Chromatography 411: 177-184 (1984)). Cka-1 (with purity> 98%) is eluted from the column in 6 M guanidine hydrochloride. Re-naturalization of the protein out of GnHCl can be carried out by various protocols (Jaenicke, R. and Rudolph, R., Protein Structure A Practical Approach, IRL Press, New York (1990)). Initially, the dialysis step is used to eliminate guanidine hydrochloride. Alternatively, the purified protein isolated from the Ni-chelate column can be attached to a second column on which a linear gradient in decrease of guanidine hydrochloride is run. The protein is allowed to renature while bound to the column, and is subsequently eluted with a buffer containing 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl, pH 7.5 and 10% glycerol. Finally, the soluble protein is dialyzed against a storage buffer containing 5 mM ammonium acid carbonate.

Example 3 Expression of Recombinant Ckß-11 in COS Cells The plasmid expression, Ckß-11 HA is derived from a pcDNAI / Amp vector (Invitrogen) which contains: 1) the SV40 origin of replication, 2) the ampicillin resistance gene, 3) the origin of replication with E. coli, 4) the CMV promoter followed by a polylinker region, an SV40 intron and the polyadenylation site. A DNA fragment encoding the complete Ckβ-11 precursor and an HA tag fused to the structure at its 3 'end is cloned into the vector polylinker region, therefore, the expression of the recombinant protein is directed under the CMV vector. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, as previously described (I. Wilson, H. Niman, R., Hiegthen, AC erenson, M. Connolly, and R. Lerner, 1984, Cell 37 , 767). Infusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows: The DNA sequence coding for Ckß-11, ATCC # 75948, is constructed by PCR using two primers: the 5d primer 5'-AAAAAGCTTGCCATGGCCCTGCTACTG-3 'contains a HindIII site followed by 18 nucleotides of the coding sequence of Ckß-11 * starting from the position minus 3 relative to the initiation codon; the sequence in 3d direction 5'-CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTATAGGTTAACTGCTGC GAC 3 'contains the complementary sequences for a Xbal site, the stop codon of the translation, the HA tag and the last 18 nucleotides of the coding sequence Ckß-11 (not including the codon of detention). Therefore, the PCR product contains a HindIII site, the coding sequence of Ckβ-11 followed by the HA tag fused within the structure, a stop codon of the translation termination next to the HA tag and a Xbal site . The DNA fragment amplified by PCR and the vector, pcDNAI / Amp, are digested with the restriction enzymes HindIII and Xbal and then ligated. The ligation mixture is transformed into the SURE strain of E. coli (Stratagene Cloning Systems, La Jolla, CA) the culture is plated on plates with ampicillin media, and resistant colonies are selected. The plasmid DNA is isolated from the transformants and examined by restriction analysis for the presence of the correct fragment. For expression of recombinant Ckβ-11, COS cells are transfected * with the expression vector by the DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the HA protein of Ckβ-11 is detected by the radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). The cells are labeled for 8 hours with! 'S-cysteine two days after transfection. The culture media are then harvested and the cells are lysed with detergent (RIPA buffer (150 M NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al, Id. 37: 767 (1984)) Both cell culture and lysing media are precipitated with an HA specific monoclonal antibody The precipitated proteins were analyzed by SDS-PAGE.

Example 4 Expression of recombinant Cka-3 in COS cells The plasmid expression, Cka-1 HA is derived from a pcDNAI / Amp vector (Invitrogen) which contains: 1) the SV40 origin of replication, 2) the resistance gene to * ampicillin, 3) the origin of replication of E. col i, 4) the CMV promoter followed with a polylinker region, an SV40 intron and the polyadenylation site. A DNA fragment encoding the complete Cka-1 precursor and an HA tag fused to the structure at its 3 'end, is cloned into the polylinker region of the vector, therefore, the expression of the recombinant protein is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, as previously described (I. Wilson, H. Niman, R., Hiegthen, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37 , 767). Infusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The construction strategy of the plasmid is described as follows: The DNA sequence encoding Cka-1, ATCC # 75947, is constructed by PCR using two primers: primer 5'-AAAAAGCTTAGAATGAAGTTCATCTCG-3 'contains a HindIII site followed by 18 nucleotides of the coding sequence of Cka-1 starting from the position minus 3 relative to the start codon; the sequence in 3d direction 5'- CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTAGGGAATCTTTCTCTT 3 'contains the complementary sequences for a Xbal site, the stop codon of the translation, the HA tag and the last 18 nucleotides of the coding sequence of Cka-1 (not including codon of detention). Therefore, the PCR product contains a HindIII site, the coding sequence of Cka-1 followed by the HA tag fused within the structure, a stop codon of the translation termination next to the HA tag and a Xbal site . The DNA fragment amplified by PCR and the vector, pcDNAI / Amp, are digested with the restriction enzymes HindIII and Xbal and then ligated. The ligation mixture is transformed into the SURE strain of E. coli (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on media plates with ampicillin and the resistant colonies are selected. The plasmid DNA is isolated from the transformant and examined by restriction analysis for the presence of the correct fragment. For expression of recombinant Ckβ-11, COS cells are transfected * with the expression vector by the DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the HA protein of Cka-1 is detected by the radiolabeling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labeled for 8 hours with 'S-cysteine two days after transfection. The culture media are then collected and the cells are used with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al, Id. 37: 767 (1984)) Cell lysate and culture media are precipitated with an HA-specific monoclonal antibody The precipitated proteins were analyzed by SDS-PAGE.

Example 5 Cloning and expression of Ckß-11 using the baculovirus expression system The DNA sequence encoding the full length Ckß-11 protein, ATCC # 75498, is amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the gene: the primer in the 5 'direction has the sequence CGCGGGATCCGCCATCATGGCCCTGCTACTGGCCCT 3 'and contains a restriction enzyme site BamHl (in bold) followed by 6 nucleotides that resemble an efficient signal for the initiation of translation in eukaryotic cells (Kosak, MJ Mol. Biol. 196: 947-950 (1987) which is just behind the first 20 nucleotides of the Ckß-11 gene (the start codon for the translation "ATG" is underlined): The primer in the 5 'direction has the sequence CGGCGGTACCTGGCTGCACGGTCCATAGG 3' and contains the site of cleavage for the restriction endonuclease Asp781 and 19 nucleotides complementary to the 3 'untranslated sequence of the Ckß-11 gene The amplified sequences are isolated from a 1% agarose gel, using commercially available equipment ("Geneclean", BIO 101 Inc. La Jolla, CA) The fragment is then digested with the endonucleases BamHI and Asp781 and then purified again on a 1% agarose gel.This fragment is designated F2. or pRG1 (modification of vector pVL941, discussed further on) is used for the expression of the Ckβ-11 protein using the baculovirus expression system (for review see: Summers, M. D. and Smith, G.E. 1987, A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus.

(AcMNPV) followed by the recognition sites for the BamH1 and Asp781 endonucleases. The polyadenylation site of simian virus (SV) 40 is used for efficient polyadenylation. For easy selection of the recombinant viruses, the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter, followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sites by viral sequences, for the cell-mediated homologous recombination, of cotransfected wild-type viral DNA. Many other baculoviral vectors could be used in place of pRG1, such as pAc373, pVL941, and pAcIMl (Luckow, V.A. and Summers, M.D .:, Virology, 170: 31-39). The plasmid is digested with the restriction enzymes BamHI and Asp781 and then dephosphorylated using calf intestinal phosphatase, by methods known in the art.

The DNA is then isolated from a 1% agarose gel using the commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, CA). This vector DNA is designated V2. F2 fragment and dephosphorylated plasmid V2 are ligated with T4 DNA ligase. The HB101 cells of E. col i are then transformed and the bacteria containing the plasmid (pBac-Ckß-11) are identified with the Ckβ-11 gene using the enzymes Ba Hl and Asp781. The sequence of the cloned fragment is confirmed by DNA sequencing. 5μg of plasmid pBac-Ckβ-11 are co-transfected with 1.0 μg of a commercially available, linearly available baculovirus ("baculovirus DNA" BaculoGold (RM) ", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. collaborators, Proc. Nati, Acad. Sci. USA, 84: 7413-7417 (1987)), 1 μg of Baculogold1RM viral DNA) and 5 μg of plasmid pBac-Ckß-11 are mixed in a sterile well of a microtiter plate which contains 50 μl of serum free Grace medium (Life Technologies Inc., Gaithersburg, MD), then add 10 μl of lipofectin plus 90 μl of Grace medium, mix and incubate for 15 minutes at room temperature. of transfection is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture dish with 1 ml of Grace's serum-free medium.The plate moves in a backward and forward oscillation to Mix the newly added solution. Then cook for 5 hours at 27 ° C. After 5 hours the transfection solution is removed from the plate and the Grace insect medium supplemented with 10% fetal calf serum is added. The plate is placed again in an incubator and the culture is continued at 27 ° C for four days. After four days the supernatant is harvested and plaque assay performed in a similar manner as described by Summers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows easy isolation of the blue-stained plates (a detailed description of a "plaque assay" can also be found in the user guide for insect cell culture and baculovirology, distributed by Life Technologies Inc., Gaithersburg, pages 9-10). Four days after serial dilution, the viruses are added to the cells and the blue stained plates are collected with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect the Sf9 cells seeded in 35 mm boxes. Four days later the supernatants of these culture boxes are harvested and then stored at 4 ° C. Sf9 cells are grown in Grace's medium supplemented with 10% FBS inactivated by heat. The cells are infected with the recombinant baculovirus V-Ckß-11 at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with the SF900 II medium minus methionine and cysteine (Life Technologies Inc. Gaithersburg). 42 hours later 5μCi of 38S-methionine and 5μCi of 3: >are addedS-cysteine (Amersham). The cells are subsequently incubated for 16 hours before they are harvested by centrifugation and the labeled proteins are "visualized by SDS-PAGE and autoradiography.

Example 6 Cloning and Expression of Cka-1 using the baculovirus expression system The DNA sequence encoding the full length Cka-1 protein, ATCC # 75497, is amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequences of the gene: The primer in the 5 'direction has the sequence 5 '-GCCGGATCCGCCATCATGAAGTTCATCTCGACATC-3' and contains a BamHl restriction enzyme site (in bold) followed by 6 nucleotides that resemble an efficient signal of translation initiation in eukaryotic cells (Kosak, MJ Mol. Biol. 196: 947-950 (1987) which is just behind the first 20 nucleotides of the Cka-1 gene (the start codon for translation "ATG" is underlined): The primer in the 3 'direction has the sequence 5' -CGCGGGTACCGGTGTTCTTAGTGGAAA- 3 'and contains the cleavage site for the restriction endonuclease Asp781 * (ep boldface) and 19 nucleotides complementary to the 3' untranslated sequence of the Cka-1 gene.The amplified sequences are isolated from a gene of 1% agarose, using commercially available equipment ("Geneclean", BIO 101 Inc. La Joila, CA). The fragment is then digested with the endonucleases Ba Hl and Asp781 and then purified again on a 1% agarose gel. This fragment is designated F2. The vector pRGl (modification of vector pVL941, discussed below) is used for the expression of the Cka-1 protein using the baculovirus expression system (for review see: Summers, MD and Smith, GE 1987, A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of Autographa californi ca nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the BamH1 and Asp781 endonucleases. The polyadenylation site of simian virus (SV) 40 is used for efficient polyadenylation. For easy selection of the recombinant viruses, the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter, followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sites by viral sequences, for the cell-mediated homologous recombination, of cotransfected wild-type viral DNA. Many other baculoviral vectors could be used in place of pRG1, such as pAc373, pVL941, and pAcIMl (Luckow, V.A. and Summers, M.D .:, Virology, 170: 31-39). The piásmido is digested with the restriction enzymes BamHl and Asp781 and then dephosphorylated using calf intestinal phosphatase, by methods known in the art.

The DNA is then isolated from a 1% agarose gel using the commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, CA). This vector DNA is designated V2.

F2 fragment and dephosphorylated plasmid V2 are ligated with T4 DNA ligase. The HB101 cells of E. coli are then transformed and the bacteria containing the plasmid (pBac-Cka-1) are identified with the Cka-1 gene using the enzymes BamHl and Asp781. The sequence of the cloned fragment is confirmed by DNA sequencing. 5μg of pBac-Cka-1 plasmid are co-transfected with 1.0 μg of a commercially available, linearized baculovirus ("BaculoGold R" baculovirus DNA, Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al., Proc. Nati, Acad. Sci. USA, 84: 7413-7417 (1987)), 1 μg of Baculogold viral DNA) and 5 μg of plasmid pBac-Cka-1 are mixed in a sterile well of a microtitre plate containing 50 μl of serum-free Grace medium (Life Technologies Inc., Gaithersburg, MD) Subsequently, 10 μl of lipofectin plus 90 μl of Grace medium are added, mixed and incubated for 15 minutes at room temperature. ambient. Subsequently, the transfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture dish with 1 ml of Grace's medium without serum. The plate moves in oscillation back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 ° C. After 5 hours the transfection solution is removed from the plate and the Grace insect medium supplemented with 10% fetal calf serum is added. The plate is placed again in an incubator and the culture is continued at 27 ° C for four days. After four days the supernatant is harvested and plaque assay performed in a similar manner as described by Summers and Smith (supra). As a modification, a Garosa gel with "Blue Gal" is used (Life Technologies Inc., Gaithersburg) which allows easy isolation of the blue-stained plates (a detailed description of a "plaque assay" can also be found in the user guide for insect cell culture and baculovirology, distributed by Life Technologies Inc. , Gaithersburg, pages 9-10). Four days after serial dilution, the viruses are added to the cells and the blue stained plates are collected with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect the Sf9 cells seeded in 35 mm boxes. Four days later the supernatants of these culture boxes are harvested and then stored at 4 ° C. Sf9 cells are grown in Grace's medium supplemented with 10% FBS inactivated by heat. The cells are infected with the recombinant baculovirus V-Cka-1 at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with the SF900 II medium minus methionine and cysteine (Life Technologies Inc. Gaithersburg). 42 hours later, 5μCi of 3bS-methionine and 5μCi of ^ S-cysteine (Amersham) are added. The cells are subsequently incubated for 16 hours before they are harvested by centrifugation and the labeled proteins are visualized by SDS-PAGE and autoradiography.

Example 7 Expression Via Gene Therapy Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. "The small pieces of tissue are placed on a wet surface of a tissue culture flask, approximately 10 pieces are placed in each flask, the flask is turned upside down, sealed, and left at room temperature overnight After 24 hours at room temperature the flask is inverted and the pieces of tissue remain fixed to the bottom of the flask and fresh medium is added (for example, Ham's F12 medium, with 10% FBS, penicillin and streptomycin). It is then incubated at 37 ° C for about a week.At this time fresh medium is added and subsequently changed every several days.After an additional period of two weeks in culture, a monolayer of fibroblasts emerges.The monolayer is trypsinized and increases in scale to higher flasks. pMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)) flanked by the long terminal repeats of Moloney murine sarcoma virus, digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase . The linear vector is fractionated on agarose gel and purified, using glass spheres. The cDNA encoding a polypeptide of the present invention is amplified using the PCR primers which correspond to the 5 'and 3' end sequences, respectively. The 5 'primer containing an EcoRI site and the 3' primer further includes a HindIII site. Equal amounts of the linear backbone of the Moloney murine sarcoma virus and the amplified EcoRI and HindIII fragment are added together in the presence of the T-DNA ligase. The resulting mixture is maintained under conditions appropriate for the ligation of the two fragments. The ligation mixture is used to transform HB101 bacteria, which are then plated onto agar containing kanamycin, in order to confirm that this vector has the gene of interest properly inserted.

The amphotropic amplicon pA317 or GP + aml2 packaging cells are grown in tissue culture to a confluent density in Dulbecco's Modified Eagle Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the medium and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles that contain the gene (the packaging cells are now referred to as producer cells). Fresh media is added to the transduced producer cells, and subsequently, the medium is harvested from a 10 cm plate of the confluent producer cells. The spent medium, which contains the infectious viral particles, is filtered through a Millipore filter to eliminate the uncoupled producer cells, and this medium is then used to infect the fibroblast cells. The medium is removed from a subconfluent plate of fibroblasts and rapidly replaced with the medium from the producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. The genetically engineered fibroblasts are then injected into the host, either alone or after being developed to confluence on Cytodex 3 microcarrier spheres. The fibroblasts now produce the protein product. Numerous modifications and variations of the present invention are possible, in light of the above teachings and, therefore, within the scope of the appended claims the invention may be practiced otherwise than as particularly described.

LIST OF SEQUENCES GENERAL INFORMATION i) APPLICANTS: HUMAN GENOME SCIENCES, INC. AND HAODONG Ll i) TITLE OF THE INVENTION: Chemokine 11 Beta-11 Human and Chemokine Alfa-l Human iii) SEQUENCE NUMBER: 16 iv) DOMICILE FOR CORRESPONDENCE: A) RECIPIENT: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C. B) STREET: 1100 New York Avenue, N.W. C) CITY: Washington D) STATE: D.C. E) COUNTRY: UNITED STATES OF AMERICA F) POSTAL CODE: 20005-3934 v) DATA OF THE CURRENT APPLICATION: A) NUMBER OF APPLICATION: TO BE ASSIGNED B) DATE OF SUBMISSION: JUNE 5, 1996 C) CLASSIFICATION: vi) DATA FROM THE PREVIOUS APPLICATION A) APPLICATION NUMBER: 08 / 460,987 and 08/464, 401 B) DATE OF SUBMISSION: JUNE 05, 1995 and JUNE 5, 1995 vii) "ATTORNEY / AGENT INFORMATION: A) NAME: GOLDSTEIN, JORGE A. B) REGISTRATION NUMBER: 29,021 C) REFERENCE NUMBER / CASE: 1488.038PC02 viii) INFORMATION FOR TELECOMMUNICATIONS: A) TELEPHONE: (202) 371-2600 B) TELEFAX: (202) 371-2540 INFORMATION FOR SEQ ID No. 1: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 297 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: cDNA xi) SEQUENCE DESCRIPTION: SEQ ID No. 1 ATGGCCCTGC TACTGGCCCT CAGCCTGCTG GTTCTCTGGA CTTCCCCAGC CCCAACTCTG 60 AGTGGCACCA ATGATGCTGA AGACTGCTGC CTGTCTGTGA CCCAGAAACC CATCCCTGGG 120 TACATCGTGA GGAACTTCCA CTACCTTCTC ATCAAGGATG GTTGCAGGGT GCCTGCTGTA 180 GTGTTCACCA CACTGAGGGG CCGCCAGCTC TGTGCACCCC CAGACCAGCC CTGGGTAGAA 240 CGCATCATCC AGAGACTGCA GAGGACCTCA GCCAAGATGA AGCGCCGCAG CAGTTAA 297 2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 2: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 98 AMINO CIDOS 5 B) TYPE: AMINO ACID C) TYPE OF HEBRA: D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: PROTEIN 0 xi) DESCRIPTION OF THE. SEQUENCE: SEQ ID No. 2: Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser -15 -10 -5 Pro Wing Pro Thr Leu Ser Gly Thr Asn Asp Wing Glu Asp Cys Cys 1 5 10 Leu Ser Val Thr Gli Lys Pro lie Pro Gly Tyr lie Val Arg Asn 15 20 25 Phe His Tyr Leu Leu He Lys Asp Gly Cys Arg Val Pro Ala Val 30 35 40 Val Phe Thr Thr Leu Arg Gly Arg Gln Leu Cys Pro Pro Wing Asp 45 50 55 Gln Pro Trp Val Glu Arg He He Gln Arg Leu Gln Arg Thr Ser 60 -65- 70 Ala Lys Met Lys Arg Arg Ser Ser 75 80 2) INFORMATION FOR SEQ ID No. 3: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 330 PAIRS OF BASES 5 B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: cDNA 0 xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 3: ATGAAGTTCA TCTCGACATC TCTGCTTCTC ATGCTGCTGG TCAGCAGCCT CTCTCCAGTC 60 CAAGGTGTTC TGGAGGTCTA TTACACAAGC TTGAGGTGTA GATGTGTCCA AGAGAGCTCA 120 GTCTTTATCC CTAGACGCTT CATTGATCGA ATTCAAATCT TGCCCCGTGG GAATGGTTGT 180 CCAAGAAAAG AAATCATAGT CTGGAAGAAG AACAAGTCAA TTGTGTGTGT GGACCCTCAA 240 GCTGAATGGA TACAAAGAAT GATGGAAGTA TTGAGAAAAA GAAGTTCTTC AACTCTACCA 300 GTTCCAGTGT TTAAGAGAAA GATTCCCTGA 330 2) INFORMATION FOR THE SEQUENCE: SEQ ID No. 4: i) CHARACTERISTICS "OF THE SEQUENCE: A) LENGTH: 109 AMINO ACIDS B) TYPE: ACID AMINO 5 C) TYPE OF HEBRA: D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: PROTEIN 0 xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 4: Met Lys Phe Be Thr Ser Leu Leu Leu Met Leu Leu Val Ser -20 -15 -10 Ser Leu Ser Pro Val Gln Gly Val Leu Glu Val Tyr Tyr Thr Ser -5 1 5 Leu Arg Cys Arg Cys Val Gln Glu Ser Val Val Phe He Pro Arg 10 15 20 Arg Phe He Asp Arg He Gln He Leu Pro Arg Gly Asn Gly CyS 25 30 35 Pro Arg Lys Glu He He Val Trp Lys Lys Asn Lys Ser He Val 40 45 50 Cys Val Asp Pro Gln Wing Glu Trp He Gln Arg Met Met Glu Val 55 • 60 65 Leu Arg Lys Arg Being Ser Thr Leu Pro Val Pro Val Phe Lys 70 75 80 Arg Lys He Pro 85 2) INFORMATION FOR SEQ ID No. 5: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 27 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 5: CCCGCATGCC AACTCTGAGT GGCACCA 27 2) INFORMATION FOR SEQ ID No. 6: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 27 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 6: CCCGGATCCC AATGCTTGAC TCGGACT 27 2) INFORMATION FOR SEQ ID No. 7: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 30 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 7: CCCGCATGCC TTCTGGAGGT CTATTACACA 30 2) INFORMATION FOR SEQ ID No. 8: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 28 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 8: CCCGGATCCG GGAATCTTTC TCTTAAAC 28 2) INFORMATION FOR SEQ ID No. 9: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 27 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 9: AAAAAGCTTG CCATGGCCCT GCTACTG 27 2) INFORMATION FOR SEQ ID No. 10: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 57 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 10: CGCTCTAGAT TAAGCGTAGT CTGGGACGTC GTATGGGTAT AGGTTAACTG CTGCGAC 57 2) INFORMATION FOR SEQ ID No. 11: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 27 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 11: AAAAAGCTTA GAATGAAGTT CATCTCG 27 2) INFORMATION FOR SEQ ID No. 12: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 54 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF SEQUENCE: SEQ ID No. 12 CGCTCTAGAT TAAGCGTAGT CTGGGACGTC GTATGGGTAG GGAATCTTTC TCTT 54 2) INFORMATION FOR SEQ ID No. 13: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 36 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 13: CGCGGGATCC GCCATCATGG CCCTGCTACT GGCCCT 36 2) INFORMATION FOR SEQ ID No. 14: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 29 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 14: CGGCGGTACC TGGCTGCACG GTCCATAGG 29 2) INFORMATION FOR SEQ ID No. 15: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 35 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 15: GCCGGATCCG CCATCATGAA GTTCATCTCG ACATC 35 2) INFORMATION FOR SEQ ID No. 16: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 27 PAIRS OF BASES B) TYPE: NUCLEIC ACID C) TYPE OF HEBRA: SIMPLE D) TOPOLOGY: LINEAR ii) TYPE OF MOLECULE: Oligonucleotide xi) DESCRIPTION OF SEQUENCE: SEQ ID No. 16: CGCGGGTACC GGTGTTCTTA GTGGAAA 27 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (20)

1. An isolated polynucleotide, characterized in that it comprises a member selected from the group consisting of: a) a polynucleotide encoding the polypeptide as described in SEQ ID No. 2; b) a polynucleotide encoding the polypeptide as described in SEQ ID No. 4; b) a polynucleotide capable of hybridizing, and which at least 70% identical to, the polynucleotide of (a) or (b); and c) a polynucleotide fragment of the polynucleotide (a), (b) or (c).

2. The polynucleotide according to claim 1, characterized in that the polynucleotide is DNA.

3. The polynucleotide according to claim 2, characterized in that it encodes the polypeptide comprising amino acids 1 to 81 of SEQ ID No. 2.

4. The polynucleotide according to claim 2, characterized in that it encodes the polypeptide comprising amino acids 1 to 87 of SEQ ID No. 4.

5. The polynucleotide according to claim 2, characterized in that it comprises a nucleotide sequence - selected from the group consisting of SEQ ID No. 1 or SEQ ID No. 2.

6. An isolated polynucleotide, characterized in that it comprises a member selected from the group consisting of: a) a polynucleotide that encodes a mature polypeptide encoded by the DNA contained in ATCC Deposit No. 75948; b) a polynucleotide encoding a mature polypeptide encoded by DNA contained in ATCC Deposit No. 75947; c) a polynucleotide capable of hybridizing, and which is at least 70% identical to, the polynucleotide of (a) or (b); and d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).

7. A vector, characterized in that it contains the DNA according to claim 2.

8. A host cell, characterized in that it is manipulated by genetic engineering with the vector according to claim 7.

9. A process for producing a polypeptide, characterized in that it comprises: expressing from said host cell according to claim 8, the polypeptide encoded by said DNA.

10. A process for producing cells capable of expressing a polypeptide, characterized in the process because it comprises the transformation or transfection of the cells with the vector according to claim 7.

11. A polypeptide, characterized in that it is selected from the group consisting of i) a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and fragments, analogs and derivatives thereof; ii) a polypeptide having the deduced amino acid sequence of SEQ ID No. 4 and fragments, analogs and derivatives thereof; iii) a polypeptide encoded by the ATCC cDNA Deposit No. 75948 and fragments, analogs and derivatives of said polypeptide; and iv) a polypeptide encoded by the ATCC cDNA Deposit No. 75947 and fragments, analogs and derivatives of said polypeptide.

12. A compound, characterized in that it is effective as an agonist for the polypeptide according to claim 11.

13. A compound, characterized in that it is effective as an antagonist against the polypeptide according to claim 11.

14. A method for the treatment of a patient in need of Ck? -ll, characterized in that the method comprises: administering to the patient a therapeutically effective amount of the polypeptide according to claim 11.

15. The method according to claim 14, characterized in that the therapeutically effective amount of the polypeptide is administered by provision to the patient, of the DNA encoding the polypeptide and expressing said polypeptide in vi.

16. A method for the treatment of a patient in need of Cka-1, characterized in that the method comprises: administering to the patient a therapeutically effective amount of the polypeptide according to claim 12.

17. A method for the treatment of a patient in need of inhibiting a chemokine polypeptide, characterized in that the method comprises: administering to the patient a therapeutically effective amount of the antagonist according to claim 13.

18. A process for diagnosing a disease or a susceptibility to a disease related to the expression of the polypeptide according to claim 11, characterized the process because it comprises: the determination of a mutation in the nucleic acid sequence encoding said polypeptide.

19. A diagnostic process, characterized in that it comprises: the analysis of the presence of the polypeptide according to claim 11, in a sample derived from a host.

20. A method for identifying compounds that bind to and activate or inhibit a receptor for the polypeptide according to claim 11, characterized in that it comprises: contacting a cell expressing on the surface thereof a receptor for the polypeptide , said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound to be selected under conditions that allow binding to the receptor; and determining whether the compound binds to and activates or inhibits the receptor, by detecting the presence or absence of a signal generated from the interaction of the compound with the receptor.