MELANOMA-ASSOCIATED PROTEIN

Abstract: This invention relates to a melanoma-associated protein (MCSP), a derivative of said protein and to means and methods for the production thereof. The invention is also directed to isolated nucleic acids coding for said melanoma-associated protein, to a method of obtaining such nucleic acid molecules, and to their expression. Furthermore, the invention is directed to uses of said protein and nucleic acid, particularly uses relating to diagnosis, prophylaxis and therapy of tumors producing the protein, such as human malignant melanoma, sarcoma and glioblastoma.

Description: Melanoma-associated Protein This invention relates to a melanoma-associated protein (MCSP), a derivative of said protein and to means and methods for the production thereof. The invention is also directed to isolated nucleic acids coding for said melanoma-associated protein, to a method of obtaining such nucleic acid molecules, and to their expression. Furthermore, the invention is directed to uses of said protein and nucleic acid, particularly uses relating to diagnosis, prophylaxis and therapy of tumors producing the protein, such as human malignant melanoma, sarcoma and glioblastoma. During the last two decades there has been considerable interest in the biology and pathophysiology of human malignant melanoma, in part, because of the poor prognosis and increasing incidence of this disease. The fatal nature of human cutaneous melanoma, which is attributable to poor response to conventional radiation and chemotherapy, has prompted a growing interest in melanoma-associated antigens (MAAs). Such proteins are expressed on melanoma cells but not on normal skin melanocytes and include antigens that are unique for melanoma, or particular stages of melanoma progression, and others that are typical for all tumors of neuroectodermal origin. Based on proven and putative biochemical and immunological characteristics MAAs may be categorized into cell substrate-interacting glycoproteins, ion transport and binding proteins, gangliosides, and receptors for growth factors. The category of cell substrate-interacting glycoproteins comprises several MAAs of relatively high molecular weight Up to today, murine monoclonal antibodies (mAb) raised against human melanoma cells or membrane preparations of such cells have been relied upon for identification and partial characterization of these antigens. Thus, human melanoma chondroitin sulfate proteoglycan (MCSP), also referred to as high molecular weight-melanoma associated antigen (HMW-MAA), has been identified with mAb 9.2.27 (Morgan et al., Hybridoma 1, 27-36 (1981)). MCSP is expressed on more than 90% of human melanoma tissues and cultures where 80 to 100 % of cells express MCSP at densities ranging from lxlO 5 to 6xl0 6 binding sites per cell (Bumol and Reisfeld, Proc. Natl. Acad. Sci. U.S.A.79, 1245-1249 (1982); Bumol et al., J. Biol. Chem. 267, 12733-12741 (1984); Mueller and Reisfeld in Encyclopedia of Human Biology (Dulbecco, ed.), pp. 957-967, Academic Press, New York (1991)). MCSP has been reported to be a unique glycoprotein-proteoglycan complex. A 250 kDa molecule is the core glycoprotein of MCSP possessing asparagine-N-linked oligosaccharides of the high mannose type. Addition of chondroitin sulfate glycosaminolycan polysyccharide chains to serine residues of me core glycoprotein converts the 250 kDa core protein to the high molecular weight proteoglycan form. The molecular mass of the mature proteoglycan containing a full complement of chondroitin sulfate chains has been estimated at 420 to 1000 kDa (Harper and Reisfeld in "Biology of Proteoglycans" (Wight and Mecham eds.), pp. 345-366, Academic Press, Orlando (1987)). The primary structure of the core protein of the rat homologue, referred to as NG2, is known from Nishiyama et al.. J. Cell Biology 114, 359-371 (1991). Proteoglycans have been implicated in growth control, involvement in adhesion, in cell-substratum interaction and cell-cell contacts (Hardingham and Fosang, FASEB J. 6, 861-870 (1992)). Thus, MCSP is found to be expressed on the melanoma cells upper surface on microspikes, consisting of 1-2 μm structures that range up to 20 μm at the cell periphery. These peripheral structures are involved in cell-cell contacts and also form complex footpads that are in contact with the substratum (Bumol et al., 1984, supra; Harper et al., J. Immunol. 132, 2096-2104 (1984); Garrigues et al., J. Natl. Cancer Inst.71, 259-263 (1986)). Adhesion plaques deposited along the cell membrane also expressed MCSP very well (Harper et al., supra; Harper and Reisfeld, J. Natl. Cancer Inst. 71, 259-263 (1983)). A possible role of this molecule in stabilizing cell-substratum interactions is suggested by the finding that mAb 9.2.27, directed against both the proteoglycan and the core protein, blocks early events of melanoma cell spreading on endothelial basement membranes, while only slightly interfering with cell adhesion. Data indicating that MCSP core protein is expressed on the cell surface in two forms, either modified by the addition of chondroitin sulfate chains or chondroitin sulfate nonmodϋϊed, suggest that glycosaminoglycan (GAG) chains may not be necessary for cell surface expression of the core protein. Hence, it seems unlikely that such a modification serves as a marker to segregate molecules on the cell surface (Harper et al., J. Biol. Chem. 261, 3600-3606 (1986)). Furthermore MCSP recognized by mAb 9.2.27 is reported to act as a co-receptor for spreading and focal contact formation in association with α4βl integrin in melanoma cells, implying a model in which MCSP communicates with α4βl integrin by an inside-out signaling mechanism (Iida et al., Cancer Research 55, 2177-2185 (1995)). MCSP also proved to be an effective target for radioimaging of tumors of melanoma patients (Oldham et al., J. Clin. Oncol. 2, 1235-1245 (1995)) and is currently used to target active-specific immunotherapy with antiidiotypic mAb, which bear the internal image of antigenic determinanis defined by anti-MCSP mAb (Kusama et al., J. Immunol. 143, 3844-3852 (1989); Chen et al., Cancer Res.53, 112-119 (1993)). Thus, conjugation of antiidiotypic mAb with a carrier and administration with an adjuvant induced humoral anti-MCSP immunity in about 60% of immunized patients with advanced melanoma (Mittelman et al., Proc. Natl. Acad. Sci. U.S.A. 89, 466-470 (1992)). Development of this anti-MCSP immunity was found to be associated with statistically significant prolongation of survival. Although some features of MCSP have been described in the literature as shown above, the precise structure of MCSP has not been previously established. In view of the pathological significance of high MCSP expression, particularly in metastatic lesions, there is a need for a better understanding of cellular signal transduction via MCSP as well as in the role of MCSP in the interaction with surrounding cells and tumor spreading. So far, a deeper insight into human malignant melanoma and tumor progression as well as eventual improvements in diagnosis, prophylaxis and therapy of neoplasms showing high MCSP expression has been significantly hampered by the inavailability of MCSP in a purified form and amino acid and nucleic acid sequence information. This lack of knowledge has particularly handicapped the search for human therapeutic agents capable of influencing tumor growth and metastatic spreading. The present invention has achieved the isolation and sequencing of DNA encoding full-length human MCSP, thus providing the amino acid sequence of human MCSP and enabling the production of MCSP, e.g. by recombinant DNA techniques. Synthesis of a complete cDNA coding for the full-length protein was extemely difficult and could not be achieved by conventional methods. The present invention for the first time enables correlations between MCSP structure and function, thereby providing e.g. means for improved diagnosis, prophylaxis and therapy of a tumor characterized by MCSP expression, e.g. a melanoma, glioma or sarcoma expressing MCSP. More specifically, the present invention relates to a purified or isolated protein designated MCSP, or a derivative thereof. As used herein before or hereinafter, the term "purified" or "isolated" is intended to refer to a molecule of the invention in an essentially pure form, said molecule being obtainable from a natural source or by means of genetic engineering. The purified protein, DNA or RNA of the invention may be useful in ways that the protein, DNA and RNA as they naturally occur are not, such as identification of compounds selectively modulating the expression or the activity of MCSP. In a preferred embodiment, the invention concerns a protein having the amino acid sequence set forth in SEQ ID NO:2, and particularly a mature protein having the amino acid sequence extending from the amino acid at position 1 (Ala) to the amino acid at position 2293 (Val). Hereinafter, such protein will be referred to as MCSP. The peptide comprising amino acids -29 to -1 of SEQ LD NO:2 represents the MCSP signal peptide. MCSP is found to be an integral membrane protein with a large amino-terminal ectodomain separated from a relatively short cytoplasmic tail by a single hydrophobic transmembrane region. Included within the scope of " isolated MCSP" or "a protein of the invention", as these terms are understood herein, is any deglycosylated, unglycosylated or glycosylated form of the protein having the amino acid sequence set forth in SEQ ED NO:2, a splice variant encoded by mRNA generated by alternative splicing of a primary MCSP-encoding transcript, and an amino acid mutant of the protein of SEQ ID NO:2. Additionally, the invention concerns an in vitro generated covalent or aggregative derivative of a protein of the invention. According to the invention, purified MCSP is essentially free of all naturally occurring substances with which it is typically found in human tissue. For example, MCSP produced by recombinant means will be free of those contaminants typically found in its in vivo physiological milieu. Purified MCSP also encompasses a protein according to the invention in recombinant cell culture. A beforementioned protein of the invention, or a derivative thereof, displays a biological profile which is qualitatively essentially identical to the profile characteristic of native MCSP, or at least a cross-section of said MCSP- profile. The biological profile in vitro and in vivo includes antigenicity, ligand binding and signal transduction. The biological profile of a protein of the invention, or a particular biological activity thereof, may be evaluated in a suitable assay employing said protein in a purified form or a host cell producing MCSP. In any case, a protein of the invention bears at least one immune epitope in common with MCSP, or mimics such epitope. Such protein is referred to as immunological equivalent of MCSP. A protein which bears at least one immune epitope in common with MCSP comprises at least eight to about eleven consecutive amino acids of SEQ ID NO:2 and is capable of cross-reacting with an antibody which is specific for native MCSP. Thus, a protein of the invention is capable of competing with native MCSP for binding to an anti-MCSP antibody, e.g. such antibody raised against melanoma cells or a membranous fraction thereof. Examples of such antibodies are mAb 9.2.27 (Bumol and Reisfeld, Proc. Natl. Acad. Sci. U.S.A. 1245-1249 (1982)), mAb 225.28 (European Patent No. 0 380607, ATCC accession no. HB 10141) and mAb 763.74 (Giacoraini et al., J. Immunol. 135, 696 (1985)). MCSP acts as a cell surface receptor for human type VI collagen. Therefore, an assay suitable for determining the ligand binding activity of a protein of the invention is an assay deteπnining the interaction between said protein of the invention and collagen VI. Such an assay is known in the art (see e.g. Stallcup et al., J. Cell Biol. 111, 3177-3188 (1990); Nishiyama and Stallcup, Mol. Biol. Cell 4, 1097-1108 (1993)) and comprises contacting a cell producing a protein of the invention with collagen VI and assessing the binding to collagen VI to said cell as compared to a suitable negative control, e.g. by imraunofluorescent staining. For example, mammalian cells which do not produce endogenous MCSP, or a homologue thereof, but are capable of secreting type VI collagen, such as B28 rat neural cells or U251MG human glioma cells, are transfected with a DNA coding for a membrane-bound protein of the invention. The transfected cells producing said protein of the invention on the cell-surface are assayed for the ability of the protein of the invention to anchor collagen VI to the cell surface. Alternatively, a ligand binding assay may be performed using purified collagen VI, advantageously attached to a solid phase, and an isolated protein of the invention. Cell surface MCSP is capable of modifying the function and/or activity of α4βl integrin (Iida et al., J. Cell Biol. 118, 431-444 (1992)). Hence, this ability of a protein of the invention may be tested in a conventional cell adhesion assay (see e.g. Iida et al., J. Cell Biol. 118, 431-444 (1992)) using suitable cells transfected with a DNA encoding a protein of the invention and producing said protein on the cell surface. Signal transduction of a protein of the invention may be studied by evaluating the collaboration between said protein of the invention and α4βl integrin in the modulation of cell spreading and focal contact adhesion according to methods available in the art, e.g. the method described by Iida et al. (Cancer Research 55, 2177-2185 (1995)). A glycosylated form of MCSP according to the invention is e.g. MCSP having a native (human) glycosylation pattern, e.g. a MCSP glycoprotein comprising asparagine N-linked oligosaccharides of the high mannose type, or a MCSP proteoglycan containing a partial or full complement of chondroitin sulfate chains, or a glycosylation variant having a glycosylation pattern which is different from that found for native MCSP. A nonglycosylated form of MCSP according to the invention may be obtained by deglycosylation of a glycosylated form of MCSP, e.g. by enzymatic removal of the glycosyl residues, or by expression of a nucleic acid encoding a protein of the invention in suitable prokaryotic cells. According to the invention, an amino acid mutant (mutein) may be a substitutional, insertional or deletional variant of a protein with the amino acid sequence set forth in SEQ ID NO:2. Contrary to a naturally occurring allelic or interspecies variant, such a mutant is characterized by the predetermined nature of the variation. Substitutions, deletions and insertions may be combined to arrive at an amino acid mutant of the invention. For example, a substitutional amino acid mutant is any polypeptide having an amino acid sequence substantially identical to the sequence set forth in SEQ ID NO:2, in which one or more residues have been conservatively substituted with a functionally-similar amino acid residue and which is capable of mimicking an MCSP epitope as described herein before. Conservative substitutions include e.g. the substitution of one non-polar (hydrophobic) residue, such as methionine, valine, leucine, isoleucine for another, substitution of one polar (hydrophilic) residue for another, such as between glycine and serine, between arginine and lysine, and between glutamine and asparagine. Substitutional or deletional mutagenesis may be employed to eliminate O- or N-linked glycosylation sites from MCSP. MCSP has 15 potential N-linked glycosylation sites, which are characterized by the occurrence of the acceptor amino acid asparagine (Asn) in the tripepetide sequence Asn-X-Thr(Ser), wherein X can be any of the twenty naturally occurring L-amino acids except possibly aspartic acid (Asp) (Hubbard and Ivatt, Ann. Rev. Biochem. 50, 555-583 (1981)). Potential O-linked glycosylation sites in the MCSP sequence are characterized in that a serine residue precedes a glycine residue. Such Ser/Gly pairs are located at positions 51/52, 178/179, 570/571, 966/967, 1020/1021, 1067/1068, 1131/1132, 1309/1310, 1355/1356, 1475/1476 and 1872 1873 in SEQ ID NO: 2. Deletions of cysteine or other labile amino acid residues may also be desirable, for example to increase the oxidative stability of a protein of the invention. As defined herein, a deletional amino acid mutant of MCSP also includes a fragment of mature full-length MCSP consisting of eight or more contiguous amino acids, i.e. eight to 2292 contiguous amino acids, of SEQ ID NO:2. According to the invention, such MCSP fragment is a preferred embodiment of a deletional mutant. Preferred are fragments of mature MCSP comprising from about ten to about hundred, particularly, from about ten to about fifty contiguous amino acids of SEQ ID NO:2. A major class of deletional mutants are those involving the transmembrane and/or cytoplasmic region of MCSP. The transmembrane domain succeeds the N-terminal extracellular domain and essentially consists of about 25 amino acids. Extending from about residue 2193 (Met) to about amino acid residue 2217 (Leu) in SEQ ID NO:2, this highly hydrophobic domain has the proper size to span the lipid bilayer of the cellular membrane. The cytoplasmic domain of MCSP follows the transmembrane domain and is the C-terminal sequence of amino acid residues approximately commencing at position 2218 (Arg) in SEQ ID NO: 2. Deletion or substitution of either or both of the cytoplasmic and transmembrane domains will facilitate recovery of a recombinant protein of the invention by reducing its cellular or membrane lipid affinity and improving its solubility in water or buffers so that detergents will not be required to maintain the protein in aqueous solution. An example of a deletional mutant involving the transmembrane and the cytoplasmic region of MCSP is the MCSP fragment with the sequence extending from amino acid 1 (Met) to amino acid 1593 (Val) in SEQ ID NO:2. Such deletional mutant and fragments thereof consisting of at least eight, particularly from about ten to about fifty consecutive amino acids of SEQ ID NO:2 are particularly preferred. Preferred proteins of the invention are mature MCSP having the amino acid sequence set forth in SEQ ID NO:2 in a glycosylated or non-glycosylated form, and a deletional variant thereof, which is a fragment of MCSP as defined above. A derivative of a protein of the invention is a covalent or aggregative conjugate of said protein with another chemical moiety, said derivative displaying essentially the same biological profile as the underivatized protein of the invention. An exemplary covalent conjugate according to the invention is a conjugate of a protein of the invention with another protein or peptide, such as a protein comprising a protein of the invention, particularly an MCSP fragment, and a carrier protein suitable for enhancing the in vivo antigenicity of said protein of the invention. A covalent conjugate of the invention further includes a protein of the invention labelled with a detectable group, e.g. a protein of the invention which is radiolabelled, covalently bound to a rare earth chelate or biotin, or conjugated to a fluorescent moiety. An aggregative derivative of a protein of the invention is e.g. an adsorption complex of said protein with a cell membrane. A protein of the invention is obtainable from a natural source, e.g. by isolation from human cells or human tissue expressing MCSP, such as human melanoma tissue, or, preferably, by chemical synthesis or recombinant DNA techniques. Also, a combination of these techniques may be used to obtain a protein of the invention. Based on the amino acid sequence information provided in SEQ ID NO:2 chemical synthesis of a protein of the invention is performed according to conventional methods known in the art In general, those methods comprise the sequential addition of one or more amino acid residues to a growing (poly)peptide chain. If required, potentially reactive groups, e.g. free amino or carboxy groups, are protected by a suitable, selectively removable protecting group. Chemical synthesis may be particularly advantageous for fragments of MCSP having no more than about 100, and usually no more than about 20 to 40, amino acid residues. The invention also provides a method for preparing a protein of the invention, said method being characterized in that suitable host cells producing the protein of the invention are multiplied in vitro or in vivo. Preferably, the host cells are transformed or transfected with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for a protein of the invention which DNA is controlled by said promoter. Subsequently, the protein of the invention may be recovered. Recovery comprises e.g. isolating the protein of the invention from the host cells or isolating the host cells comprising the protein, e.g. from the culture broth. Suitable host cells include eukaryotic cells, e.g. animal cells, plant cells and fungi, and prokaryotic cells, such as gram-positive and gram-negative bacteria, e.g. E. coli. As used herein, in vitro means ex vivo, thus including e.g. cell culture and tissue culture conditions. An amino acid mutant, as defined hereinbefore, may be produced e.g. from a DNA encoding a protein of SEQ ID NO:2, which DNA has been subjected to site-specific in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids. While the site for introducing an amino acid variation is predetermined, the mutation per se need not be predetermined, but random mutagenesis may be performed at the target codon or region. For example, substitutional, deletional and insertional variants are prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of the protein of the invention. Alternatively, mutants of the invention may be prepared by chemical synthesis using methods routinely employed in the art A transmembrane and/or cytoplasmic deleted or substituted amino acid mutant of the invention can be produced directly in recombinant cell culture or as a fusion with a signal sequence, preferably a host-homologous signal. For example, in constructing a procaryotic expression vector, the transmembrane and the cytoplasmic domains are deleted in favor of the bacterial alkaline phosphatase, or heat stable enterotoxin π leaders, and for yeast the domains are substituted by yeast invertase, alpha factor or acid phosphatase leaders. In mammalian cell expression the transmembrane and the cytoplasmic domains may be replaced with a mammalian cell viral secretory leader. The advantage of a variant lacking both the transmembrane and the cytoplasmic region is that it is capable of being secreted into the culture medium. A protein of the invention may also be derivatized in vitro according to conventional methods known in the art A protein of the invention, or a derivative thereof, may be used, for example, as immunogen, e.g. to raise MCSP specific immunoreagents, as immunoreagent in a drug or ligand screening assay, or in a purification method, such as affinity purification of a binding ligand. A protein of the invention, or a derivative thereof, suitable for in vivo administration and capable of competing with endogenous MCSP for an endogenous ligand, e.g. collagen VI, is envisaged as therapeutic agent. The invention also relates to the use of a protein of the invention, or a derivative thereof, for the generation of a monoclonal or polyclonal antibody, which specifically binds to MCSP. Such anti-MCSP antibody is intended to include immune sera. Particularly useful for this purpose is a MCSP fragment consisting of at least eight or more, preferably eight to about twenty, consecutive amino acids of MCSP of SEQ ID NO:2. The antibodies raised against a protein of the invention may react with a non-glycosylated or glycosylated form of MCSP, or both. Monoclonal and particularly polyclonal anti-MCSP antibodies generated against a protein of the invention may be employed as immunoreagents to detect tumor-associated MCSP expression, e.g. in the diagnosis of human malignant melanoma or in the monitoring of melanoma progression and treatment For example, such antibodies are suitable for MCSP detection in fixed paraffin embedded melanoma lesions. The antibodies are produced in a mammal, e.g. mouse, rat goat or rabbit according to methods well-established in the art For the generation of anti-MCSP antibodies to be used as immunoreagents it is advantageous, if the protein of the invention used as antigen does not comprise a glycosylation site or any part of the transmembrane domain of MCSP. Particularly useful as immunoreagent are antibodies raised against a peptide of the invention which represents a single MCSP determinant The invention also relates to the use of a suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof, as a vaccine, and to a method of vaccinating a human, comprising administration of a suitably immunogenic protein of the invention, or a suitably immunogenic covalent conjugate thereof, to said human. Such a method is intended to also refer to a method of inducing an anti-tumor response in a human comprising administration of a suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof. A suitably immunogenic protein of the invention, or a suitably immunogenic derivative thereof, is capable of inducing an anti-MCSP response in vivo. A vaccine according to the invention is applicable in the prophylactic and therapeutic treatment of patients having a disposition for or suffering from an MCSP-expressing tumor. As mentioned above, MCSP is a suitable target for active immunotherapy of melanoma, because it is expressed by a high percentage of melanoma lesions involved in metastatic spreading. Thus, a suitably immunogenic protein of the invention, or a derivative thereof, is e.g. a useful agent for the control, treatment or adjuvant treatment of a MCSP-expressing tumor, e.g. melanoma. More specifically, a suitably immunogenic protein of the invention, or a derivative thereof, can be successfully employed e.g. to cause tumor regression and/or prevent tumor recurrence of early stage melanoma patients remaining at risk for metastatic disease after surgery for the primary lesion. Such protein or derivative can be "tailor-made" to bear or mimic a specific determinant of MCSP. Preferred for use as a vaccine is recombinant MCSP of SEQ ID NO:2, or a fragment thereof consisting of at least eight or more, preferably from eight to about fifty, consecutive amino acids of SEQ ID NO:2. Advantageously, a peptide to be used as vaccine lacks a glycosylation site and consists of eight to about fourty contiguous amino acids of the extracellular domain of MCSP. Said N-terminal domain consists of about 2192 amino acids and extends from the amino acid residue at position 1 to approximately the amino acid residue at position 2192 in SEQ ID NO:2. The preferred limitation on fragment size is primarily due to the size and purity limitations on synthetic polypeptide imposed by current technologies. Particularly preferred for use as a vaccine are the fragments accentuated above. Inducement of an appropriate T-cell dependent (memory) response on vivo administration may demand enhancement of the immunogenicity of the protein of the invention, e.g. by conjugation of said protein to a carrier protein, the presence of an adjuvans or expression by vehicles suitable for life vaccination, such as viruses, bacteria or autologous antigen presenting cells. The induction of an anti-MCSP immune response following vaccination may be analyzed according to methods known in the ait, e.g. by determination of the anti-MCSP antibody litre in a body fluid of a vaccinated patient e.g. serum, by means of an enzyme-linked immunoabsorbent-type assay (ELISA). As carrier protein component a suitably immunogenic conjugate of the invention may comprise any carrier protein useful in humans, e.g a non-toxic, nonpyrogenic, water soluble, pharmaceutically acceptable carrier protein, preferably such a protein having exposed amino groups. Suitable as a carrier protein component is any proteinaceous molecule containing highly immunogenic promiscuous T-cell epitopes which will bind to a broad range of polymorphic HLA class I and class π gene products, e.g. a microbial, particularly a bacterial, protein, polypeptide or oligopeptide. Particularly preferred is a covalent conjugate of the invention comprising an above captioned MCSP fragment and a carrier protein component obtainable from a bacterial toxin, e.g. tetanus toxin (see e.g. B. Bizzini in Bacterial Vaccines, Academic Press, 1984: Tetanus, pp. 38-68) or diphteria toxin, which protein component is devoid of toxin activity, but retains the antigenic properties particular to the toxin, e.g. potent immunogenicity, e.g. a mutant diphteria toxin devoid of toxin activity (Uchida et al., J. Pharma Biol. Chem. 218, 3838-3844 (1973)). Such non-toxic carrier protein component is obtainable e.g. by detoxification of the toxin, e.g. in case of tetanus toxin, or by mutation, e.g. in case of diphteria toxin. Diphteria toxin is obtainable from culture supernatants of Corynebacterium diphteriae PW8 according to the method disclosed by R.K. Holmes, Infect. Immun. 12, 1392 (1975). The preferred carrier protein to form a covalent conjugate of the invention is the mutant diphteria toxin CRM197. CRM197 is an atoxic protein which crossreacts immunologically with diphteria toxin and is obtainable fromm culture supernatants of C. diphteriae C7 (R.K.. Holmes, supra; U.S. Patent No. 4,925,792, which are incorporated herein by reference). The CRM197 protein has the same molecular weight as the diphteric toxin and is composed of a fragment B which is identical as to its function and structure to those of the toxin, and of a fragment A, which is nontoxic and differs from the original fragment by one amino acid. The carrier protein may be covalently attached to a protein of the invention involving a functional group thereof. The coupling reaction is performed according to methods known in the art in such a way that protein aggregation is avoided. Alternatively, the MCSP-caπier protein conjugate may be produced as a fusion protein by recombinant means. The invention also relates to an immunogen for use in a mammal comprising a suitably immunogenic protein of the invention, preferably an above specified prefeired peptide of the invention, or a derivative thereof. Preferred is such immunogen comprising in a single protein such peptide according to the invention and a carrier protein, as described above. The invention also concerns pharmaceutical compositions comprising a protein according to the invention. In particular, the invention relates to a pharmaceutical composition comprising an above-specified peptide of the invention in a suitably immunogenic form, e.g. an above-specified preferred peptide of the invention covalently attached to an appropriate carrier protein, as decribed above. The pharmaceutical compositions comprise, for example, a therapeutically effective amount of a protein of the invention in a suitably immunogenic form together or in admixture with pharmaceutically acceptable, inorganic or organic, solid or liquid carriers. Preferred are pharmaceutical compositions additionally comprising an adjuvant i.e. an agent further increasing the immune response. Possible adjuvants are Freund's complete adjuvant (emulsion of mineral oil, water, and mycobacterial extracts), Freund's incomplete adjuvant (emulsion of water and oil only), mineral gels, e.g. aluminium hydroxide gels, surface active substances such as lysolecithin, polyanions, peptides, BCG (Bacillus Calmette-Guerin), etc.. Particularly preferred are pharmaceutical compositions comprising a suitably immunogenic conjugate of the invention and MF59 (international patent application WO 90/14837) as adjuvant, and, optionally, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- [ 1 ,2-dipalmitoyl--f«-glycero-3-(hydroxyphosphoryloxy]ethylamide (MTP-PE; international patent application WO 90/14837). Preferred are pharmaceutical compositions for parenteral application. Compositions for intramuscular, subcutaneous or intravenous application are e.g. isotonic aqueous solutions or suspensions, optionally prepared shortly before use from lyophilized or concentrated preparations. The pharmaceutical compositions may be sterilized and contain adjuvants e.g. for conserving, stabilizing, wetting, emulsifying or solubilizing the ingredients, salts for the regulation of the osmotic pressure, buffer and/or compounds regulating the viscosity, e.g. sodium carboxycellulose, dextran, polyvinylpyrrolidine or gelatine. They are prepared by methods known in the art, e.g. by conventional mixing, dissolving or lyoprtilizing, and contain from approximately 0.01% to approximately 50% of active ingredients. The compositions for injections are processed, filled into ampoules or vials, and sealed under aseptic conditions according to methods known in the art For example, owing to the solubility in aqueous solutions a conjugate of the invention may be formulated as a "two vial system" with an above adjuvant e.g. MF59-0. Preferred is a pharmaceutical composition comprising an above-captioned conjugate of the invention suitable for intramuscular administration in a depot formulation together with an adjuvant Also preferred is a pharmaceutical composition comprising a suitably immunogenic protein of the invention, preferably a conjugate of the invention, which is appropriate for mucosal application (H.F. Staats et al., Current Opinion in Immunology 6, 572-583 (1994)), or a stabilized pharmaceutical composition that can be swallowed for oral immunization. The specific mode of administration and the dosage will be selected by the attending physician taking into account the particulars of the patient, state and type of the disease to be treated, and the like. Furthermore, a protein of the invention, or a derivative thereof, can be used for the qualitative and quantitative determination of antibodies directed against MCSP. This is especially useful for the detection of an anti-MCSP immune response induced by an anti-melanoma vaccine, such as an antiidiotypic antibody bearing me internal image of MCSP, particularly such antibody disclosed in European Patent Application EP-A-0428 485, melanoma cells, or membraneous fractions thereof, melanoma cell lysates, or a suitably immunogenic protein of the invention. For instance, a protein of the invention, or a derivative thereof according to the invention, can be used in any of the known immunoassays which rely on the binding interaction between the idiotopes of the anti-MCSP antibody and said protein of the invention. Examples of such assays are radio-, enzyme, fluorescence, chemiluminescence, immunoprecipitation, latex agglutination, and hemagglutination immunoassays. This invention also concerns test kits for the qualitative and quantitative determination of antibodies directed against MCSP comprising a protein of the invention and/or derivatives thereof and, optionally, adjuncts. In a further aspect the present invention relates to a nucleic acid (DNA, RNA) comprising an isolated, preferably recombinant nucleic acid (DNA, RNA) coding for a protein of the invention, or a fragment of such a nucleic acid consisting of at least 14 nucleotides. According to the invention, isolated MCSP-encoding nucleic acid of the invention includes a MCSP-encoding nucleic acid present in other than in the form or setting in which it is found in nature, thus embracing such nucleic acid in ordinarily MCSP expressing cells, where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature. The MCSP gene maps to human chromosome 15. In particular, the invention provides a purified or isolated DNA molecule encoding a protein of the invention, or a fragment of such DNA suitable for use as a screening probe as specified hereinafter. By definition, such a DNA comprises a coding single stranded DNA, a double stranded DNA consisting of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself. Preferred is a DNA coding for an above protein of the invention herein identified as being preferred, or a fragment of said DNA. Preferred is a DNA coding for the mature protein having the amino acid sequence set forth in SEQ ID NO:l, particularly a DNA having substantially the nucleotide sequence set forth in SEQ ID NO: 1, or a DNA coding for a fragment of said protein consisting of at least 14 consecutive amino acids of the amino acid sequence set forth in SEQ ID NO:l excluding the DNA with the sequence extending from bp 4867 to bp 7898 in SEQ ID NO:l and the DNA with the sequence extending from bp 4858 to 5357 in SEQ ID NO:l, respectively. Particularly preferred is a DNA coding for mature MCSP set forth in SEQ ID NO:l, or coding for a MCSP fragment which is accentuated above, e.g a DNA coding for the MCSP fragment extending from amino acid 1 to amino acid 1593 in SEQ ID NO:l, or a portion of said fragment It is envisaged that a nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such modified sequences can be used to produce a mutein having an amino acid sequence differing from the sequence of MCSP found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins. Given the guidance of the present invention, a nucleic acid of the invention is obtainable according to methods well known in the art The present invention further relates to a process for the preparation of such nucleic acid. For example, a DNA of the invention is obtainable by chemical synthesis, by recombinant DNA technology or by polymerase chain reaction (PCR). A suitable method for preparing a DNA of the invention may e.g. comprise the synthesis of a number of oligonucleotides, their amplification by PCR methods, and their splicing to give the desired DNA sequence. Preparation of a DNA of the invention, or a fragment thereof by recombinant DNA technology may involve screening of a suitable cDNA or genomic library. A suitable library is commercially available, e.g. a library employed in the Examples, or can be prepared from human melanoma tissue samples, cell lines and the like. After screening the library, e.g. with a DNA including substantially the entire MCSP coding region or a suitable oligonucleotide (probe) based on a said DNA, positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding complete MCSP (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNA sequences and deduced amino acid sequence provided herein. In order to detect the presence or any abnormality of endogenous MCSP genetic screening may be carried out using a nucleotide sequence of the invention as hybridization probe. Also, based on the nucleic acid sequences provided herein antisense-type therapeutic agents may be designed. In addition to being useful for the production of an above mentioned recombinant protein of the invention, a nucleic acid of the invention is useful as probe, thus e.g. enabling those skilled in the art to identify and/or isolate nucleic acid encoding MCSP or a novel non-human homologue thereof. Such probe according to the invention may be unlabeled or labeled with a chemical moiety suitable for ready detection. As a screening probe, there may be employed a DNA or RNA comprising substantially the entire coding region of MCSP, or a suitable oligonucleotide probe based on said DNA. A suitable oligonucleotide probe (for screening involving hybridization) includes a single stranded DNA or RNA that has a sequence of nucleotides that comprises at least 14, preferably at least about 20 to 30, contiguous bases that are the same as (or complementary to) any 14 or more contiguous bases set forth in SEQ ID NO:l. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are rninimized. Examplary probes are the oligonucleotides with the sequences set forth in SEQ ID NOs. 14 to 19. For example, a method suitable for identifying a nucleic acid encoding MCSP, or a novel non-human homologue thereof, comprises contacting a sample comprising MCSP candidate DNA or RNA with a nucleic acid probe described above, and identifying nucleic acid(s) which hybridize (s) to that probe. In particular, nucleic acid according to the invention is useful e.g. in a method for determining the presence of MCSP-mRNA, said method comprising hybridizing the DNA (or RNA) encoding (or complementary to) a protein of the invention, or a fragment of said DNA, to test sample nucleic acid and determining the presence of the desired mRNA, or amplifying, e.g. by PCR, MCSP-RNA using MCSP specific oligonucleotide primers derivable from a nucleic acid sequence provided herein. This method may be employed in tumor diagnosis, e.g. for localization of MCSP mRNA in a tumor, particularly primary melanomas or metastatic lesions of malignant melanoma. For example, specific in situ hybridization signals for MCSP mRNA expression obtained with antisense RNA probes according to the invention are clearly associated with cells obtained from a metastatic lesion of malignant melanoma. Only non-specific background signals are found with tumor infiltrating cells. Hybridization with sense control RNA yields only non-specific background signals. The DNA encoding a protein of the invention can be incorporated into vectors for further manipulation. Furthermore, the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of the above mentioned DNAs. A hybrid vector of the invention comprises an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restriction sites. Preferably, the hybrid vector of the invention comprises an above described nucleic acid insert operably linked to an expression control sequence, in particular those described hereinafter. Vectors typically perform two functions in collaboration with compatible host cells. One function is to facilitate the cloning of the nucleic acid that encodes the protein of the invention, i.e. to produce usable quantities of the nucleic acid (cloning vectors). The other function is to provide for replication and expression of the gene constructs in a suitable host either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors). A cloning vector comprises the DNAs as described above, an origin of replication or an autonomously replicating sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector additionally comprises expression control sequences essential for the transcription and translation of the DNA of the invention. Thus an expression vector refers to a recombinant DNA construct such as a plasmid, a phage, recombinant virus or other vector that upon introduction into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and/or prokaryotic cells. Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be amplified by insertion into the host genome. However, the recovery of genomic DNA encoding MCSP is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise MCSP DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component Advantageously, expression and cloning vectors according to the invention contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media. Since the amplification of the vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid. Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up MCSP nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes confering resistance to G418 or hygromycin. The mammalian cell transfectants are placed under selection pressure which only those transfectants are uniquely adapted to survive which have taken up and are expressing the marker. Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to MCSP nucleic acid. Such promoter may be inducible or constitutive. The promoter is operably linked to DNA encoding a protein of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Promoters suitable for use with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, ϋiereby enabling the skilled worker operably to ligate them to DNA encoding a protein of the invention, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding MCSP. MCSP gene transcription from vectors in mammalian host cells may be controlled by promoters compatible with the host cell systems, e.g. promoters derived from the genomes of viruses. Suitable plasmids for expression of the protein of the invention in eukaryotic host cells, particularly mammalian cells, are e.g. cytomegalovirus (CMV) promoter-containing vectors, RSV promoter-containing vectors and SV40 promoter- containing vectors and MMTV LTR promoter-containing vectors. Depending on the nature of their regulation, promoters may be constitutive or regulatable by experimental conditions. Transcription of a DNA encoding a protein according to the invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Construction of vectors according to the invention employs conventional ligation techniques. The various DNA segments of the vector DNA are operatively linked, i.e. they are contiguous and placed into a functional relationship to each other. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a manner known in the art Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing MCSP expression and function are known to those skilled in the art Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting, e.g. to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), in situ hybridization, using an appropriately labelled probe based on a sequence provided herein, binding assays, immunodetection and functional assays. The invention further provides host cells capable of producing a protein of the invention and including heterologous (foreign) DNA encoding said protein. The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g. those mentioned above, that are transformed or transfected with an appropriate expression vector. A protein of the invention may also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein (s) encoded by the DNA of the invention is (are) expressed. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-prositive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5α and HB 101, or Bacilli. Further host cells suitable for MCSP encoding vectors include eukaryotic microbes such as filamentous fungi or yeast e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect amphebian and vertebrate cells, particularly mammalian cells. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. The host cells referred to in this application comprise cells in in vitro culture as well as cells that are within a host animal. Advantageously, a host cell of the invention does not produce endogenous MCSP or a homologue thereof. Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency. Host cells are transfected or transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique, by electroporation or by lipofectin-mediated. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used. (See, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press). A DNA of the invention may also be expressed in a non-human transgenic animal, particularly a transgenic warm-blooded animal, and in non-human transgenic tumor cells. Methods for producing a transgenic animal, including mouse, rat, rabbit, sheep and pig, are known in the art and are disclosed, for example, by Hammer et al. (Nature 315, 680-683, (1985)). For instance, an expression unit including a DNA of the invention coding for MCSP together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, or in tumor cells. Introduction may be achieved, e.g. by microinjection. Integration of the injected DNA is detected, e.g. by blot analysis of DNA from suitable tissue samples. It is preferred that the DNA be incorporated into the germ line of the animal, so that it is passed to the animal's progeny. Transgenic tumor cells are introduced into a suitable animal. Furthermore, a knock-out animal may be developed by introducing a mutation in the endogenous MCSP-homologue, thereby generating an animal, which does not express the functional MCSP-homologue gene anymore. For example, in a rat the NG2 gene may be knocked out A mutated or nonmutated MCSP gene is introduced into the knock-out animal. Expression of human counterpart MCSP on a homologous gene knock-out background has the unique advantage of excluding differences in efficacies of a potential drug on the given protein (in this case MCSP) caused by species-specific sequence differences in said protein. In a further aspect the invention relates to an assay for identifying a compound which is capable of interacting with MCSP, comprising contacting cells containing a heterologous DNA encoding a protein of the invention and producing said protein with at least one compound to be tested for its ability to interact with MCSP, and analysing cells for a difference in ligand binding or signal transduction. Suitable analysing methods are known in the art, or may be readily designed based on the known methods and the guidelines provided herein. Preferably, the heterologous DNA comprises substantially the entire coding region. The result obtained in such assay is compared to an assay suitable as a negative control. Assay methods generally require comparison to various controls. A change in MCSP activity or function is said to be induced by a test compound if such an effect does not occur in the absence of the test compound. An effect of a test compound on a protein of the invention is said to be mediated by said protein if this effect is not observed in cells which do not produce said protein. For example, by interacting with MCSP compounds may affect melanoma cell growth and spreading, cell-adhesion including cell-substratum interaction and cell-cell contact and MCSP related signal transduction, thus being potential anti-tumor drugs. An assay as described above is suitable to identify a compound which is capable of inhibiting the binding of collagen VI to MCSP. The invention particularly relates to the specific embodiments (proteins, nucleic acids, methods for the preparation and uses thereof) as described in the Examples which serve to illustrate the present invention, but should not be construed as limitation thereof. Example 1: Isolation of MCSP cDNA A radiolabeled approximately 4.0 kb cDNA encoding the carboxyl-terminus of the rat NG2 transcript (Gi l, cf. Fig. 2 in Nishiyama et al., J. Cell Biol. 114, 359-371 (1991), is used to screen a λgtl 1 human melanoma cDNA library prepared from RNA extracted from the M21 human melanoma cell line (Clontech Laboratories, San Francisco, CA) for MCSP candidate cDNA clones. An initial screen of recombinant phages containing dT-primed cDNA yields several NG2 reactive reactive clones. Recombinant phages (5x10^) are plated at a density of 4x 10 4 plaques per 150 mm petri dish and propagated for 12 hrs at 37°C in the Y 1090r E. coli host strain. Phage DNA is transferred to nitrocellulose filters, denatured in 0.5 N NaOH, 1.5 mM NaCl and subsequently neutralized in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl. Non-specific nucleic acid binding sites are blocked in a prehybridization medium consisting of 2xSSC (SSC: 150 mM sodium chloride, 15 mM sodium citrate) and 50 μg/ml denatured, sheared salmon sperm DNA at 55°C for 2 hrs. Hybridization reactions are performed under the same conditions for 8 to 12 hrs in the presence of 10 ng/ml of Gl 1 NG2 cDNA fragment (supra) radiolabeled with 32 P adCTP to a specific activity of 4x10 8 cpm/μg by random priming (Sam brook et al, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Following hybridization, the filters are washed in 2xSSC at 55°C and exposed to XAR film (Eastman Kodak) for autoradiography. This screen yields seven NG2 reactive clones, including one clone designated XM3.1, containing 3.1 kb of cDNA. Nucleotide sequence analysis of this isolate by the chain termination method of Sanger et al. (Sambrook et al., supra) indicates an open reading frame encoding 700 carboxyl-terminal amino acid residues with approximately 79% homology to the NG2 protein. The 3.1 kb isolate of the λM 3.1 clone (supra) is utilized as probe in Northern analysis of polyadenylated RNA extracted from human melanoma cell lines expressing the mAb 9.2.27-reactive antigen. RNA gel blotting and hybridization is done by size-fractionation of 2 μg of polyadenylated RNA on 1.2% formaldehyde-agarose gels and transferred onto nytran. Prehybridization and hybridization conditions are as described above. Transcript sizes are estimated by comparison with size standards (Gibco-Bethesda Research Laboratories). Hybridization to an 8.0 kb transcript present in M21 melanoma cells (Bumol et al., J. Biol. Chem. 267, 12733-12741 (1991)) and UAC 903 melanoma cells is observed, said transcript being similar in size to the NG2 transcript. The UAC903 cell line expresses approximately five-fold more MCSP transcript than the M21 cell line, an observation that is consistent with a similar elevation in the level of MCSP core protein produced in these cell lines. The cDNA fail to hybridize to RNA from the mAb 9.2.27-unreactive RAJI lymbhoblastoid cell line (Dierich et al., J. Immunol. 112, 1766-1773 (1974)). A 500 bp fragment from the 5' end of λM3.1 (extending from bp 4858 to 5357 in SEQ ID NO:l) is employed as a probe to screen 5 x 10 5 clones from two independent λgtl 1 cDNA libraries derived from the human melanoma cell line M21 for overlapping cDNA clones extending further upstream. Exhaustive screening of four independent melanoma cell cDNA libraries with XM3.1 fails to identify clones extending further upstream of the 5' end of λM3.1. The impossibility to obtain a complete cDNA strand is probably due to MCSP RNA secondary structure. Therefore, a strategy is developed that allows to obtain small overlapping cDNA clones. cDNA synthesis for sequence determination of the entire coding sequence of MCSP core protein is accomplished by polymerase chain reaction (PCR) using a wide range of different primers (see Table 1) and a variety of different RNA denaturation conditions to secure that suitable conditions for each individual portion are present in a sample. Thus, cDNA fragments are obtained which are suitable for PCR amplification. Experimental details for the generation of PCR-amplified cDNA clones are as follows: RNA is prepared from A375-Met human melanoma cells (Kozlowski et al., J. Natl. Cancer Inst 72, 913-917 (1984); cell line A 357 is obtainable from the American Type Culture Collection (ATCC) under accession no. ATCC CRL 1619) by the acid guanidinium thiocyanate phenol-chloroform method (Chomzynski and Sacchi, Anal. Biochem. 162, 156 (1987)). Polyadenylated RNA is obtained using a Qiagen Oligotex mRNA preparation kit (Diagen, Hilden, Germany). First strand cDNA is prepared with an M-MuLV Reverse Transcriptase Kit (Life Technologies, Gaithersburg, MD) and either MCSP sequence-specific oligonucleotides, oligo (dT) or random primers in parallel samples. Generally, the best results are obtained with MCSP sequence-specific oligonucleotides (cf. Table 1, infra). On several occasions, the primary products are reamplified with a nested 5' primer to improve specificity. PCR amplifications are done with the primers indicated in Table 2 applying standard protocols (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer Verlag, Berlin (1992)). Taq, Pwo (Boehringer Mannheim, Germany) or Pfu DNA polymerase (Stratagene, La Jolla, CA) are applied as thermostable polymerases. Anchored PCR is performed after dG tailing of cDNA (Pluschke et al., Eur. J. Immunol. 21, 2749-2754 (1991)). Prior to tailing, first strand cDNA is treated with RNAse H (Life Technologies) and purified with a Glass MAX DNA isolation spin cartridge system (Life Technologies). cDNA is dG-tailed using the Deoxynucleotidyl Terminal Transferase kit (Life Technologies). Oligonucleotides are obtained from Microsynth (Windisch, Switzerland). PCR amplification products of the expected size are isolated from 1% agarose gels and are introduced into plasmids pBluescript KS (Stratagene) or pGEM-1 (Promega, Madison, WI) either by blunt-end cloning into the Hinc π site (Table 1) or by directional cloning (Table 2). Double-stranded plasmid DNAs are sequenced directly with the Sequenase kit (US Biochemical, Cleveland, OH). Sequence data are processed with the aid of a GCG Wisconsin Software Package (Genetics Comuting Group, Madison, WI). Table 1: Clones used for establishing the MCSP primary nucleotide sequence Clone anl P10, SEQ ID NO: 13 oligo dC an38 P6, SEQ ID NO: 15 oligo dC an2 P4, SEQ ID NO: 17 oligo dC an44 4213- BA50, SEQ ID NO:18 BA51, SEQ ID NO:19 4889 oligo dC 2) oligo dC λM3.1 4866- derived from λgtl 1 cDNA 7898 library ' "pos." designates length and bp positions of the respective clone with reference to the MCSP nucleotide sequence set forth in SEQ ID NO:l. 2 ^ after dG tailing of cDNA In a first step, anchored PCR is used to obtain a clone designated an44 (Table 1) , that extends 5' of λM3.1. After dG tailing of cDNA, oligo dC is applied as 5' primer and a sequence corresponding to the 5' end of λM3.1 is used as 3' anti-sense primer in this amplification. Subsequently, a series of seven additional cDNA clones are generated by PCR to cover the entire MCSP coding sequence (see Table 1, infra). Anchored PCR with oligo dC 5' primers are applied for the amplification of three of these overlapping clones (an2, an38 and anl; Table 1), while conventional PCR using 5' sense primers that correspond to rat NG2 sequences are employed for the generation of the remaining four clones (ra23, ra4, ral and ra25; Table 1). Taq is used as DNA polymerase. Sequences of the 3' anti-sense primers used for the generation of these PCR clones are complementary to the 5' end of the respective previous clones. In five cases (cf. Table 1), the primary products are reamplified with a nested 5' primer to improve specificity. Nucleotide sequences derived from this first series of PCR clones and from the λgtl 1 clone are reconfirmed by analyzing a second set of independently derived overlapping PCR clones (Table 2). Discrepancies which are probably caused by mistakes introduced by PCR amplification (Keohavong and Thilly, Proc. Natl. Acad. Sci. USA 86, 9253 (1989)), are resolved by further analyzing independently-derived PCR clones (Table 2). Table 2: Clones used for reconfirming the MCSP primary sequence Clone pos. ^ primer H10/H4 1 - RMP16, SEQ ID NO:20 637 RMP19, SEQ ID NO:21 G3/G4/ 612 - RMP17, SEQ ID NO:22 G104 1197 RMP20, SEQ ID NO:23 F2/F3 1173 - RMP11, SEQ ID NO:24 2241 RMP18, SEQ ID NO:25 E9/ 2221 - RMP9, SEQ ID NO:26 E910 3316 RMP10, SEQ LD NO:27 D3/D23 3297 - RMP8, SEQ ID NO:28 4199 RMP12, SEQ ID NO:29 C2/C20 3757 - RMP14, SEQ ID NO:30 5137 RMP15, SEQ ID NO:31 B1/B12 5018 - RMP3, SEQ ID NO:32 B36 5869 RMP4, SEQ ID NO:33 Al/All 5848 - RMP1, SEQ ID NO:34 7216 RMP2, SEQ ID NO:35 " "pos." designates length and bp positions of the respective clone with reference to the MCSP nucleotide sequence set forth in SEQ ID NO: 1. The complete coding sequence of the MCSP core protein and the deduced amino acid sequence are shown in SEQ ID NO:l. An open reading frame coding for 2322 amino acids is found. The 3 'untranslated region consists of 926 nucleotides. The first 29 amino acids (amino acids -29 to -1; SEQ ID NO:l) represent a putative signal sequence, which is only 48% identical with that of NG2. The subsequent stretch of 18 amino acids is 89% identical. A hydrophobic segment of 25 consecutive amino acid residues near the carboxy terminus (amino acid residues 2193-2217, SEQ ID NO:2) is followed by several basic arginine and lysine residues and thus meets the criteria for a transmembrane domain. Thus, the deduced amino acid sequence of the MCSP core protein predicts an integral membrane protein comprising a large extracellular domain separated from a relatively short cytoplasmic tail (75 amino acids) by a single hydrophobic transmembrane region of 25 amino acids. The large extracellular domain of MCSP spanning 2192 amino acids can be roughly divided into three structural domains: an amino- terminal domain (amino acids 1-611, SEQ ID NO:l) containing eight cysteines and three serine glycine pairs; a cysteine-free, a serine/glycine rich domain (amino acids 612-1561, SEQ ID NO:l) including seven such potential attachment sites for glycosaminoglycans; and a third structural domain (amino acids 1562 to 2192, SEQ ID NO:l) witii only two cysteines and one serine/glycine pair. The first structural domain (amino acids 1 to 611), which is approximately 82 % structurally homologous to the corresponding domain in the rat NG2 proteoglycan, contains three of the 15 potential N-linked glycosylation sites. This domain also appears to have a compact configuration, since it contains eight of the ten cysteines of the entire ectodomain, i.e. four potential disulfide bridges in a region spanned by 611 amino acids. A key feature of the second structural domain of the MCSP ectodomain (amino acids 612 to 1561; SEQ ID NO:l) is its lack of cysteines in a region spanning 950 amino acids; however, this domain contains seven of the eleven serine/glycine pairs of the MCSP extracellular domain, which can serve as potential chondroitin sulfate attachment sites; however, the signal sequence SerGlyXGly for glycosaminoglycans (GAG) occurs only once (amino acids 1308-1340, SEQ ID NO:l). Six of the 15 potential N-linked MCSP glycosylation sites are found in this domain, which is 79% structurally homologous with its counterpart in the rat NG2 proteoglycan. The third structural domain of MCSP encompassing 630 amino acids (amino acids 1562 to 2192, SEQ ID NO:l) is approximately 75% homologous in structure with the corresponding domain of NG2. This domain consists of two cysteines, separated by 105 amino acids and likely forms a disulfide bridge. The domain has only one potential GAG attachment site indicated by one serine/glycine pair and contains six of the 15 potential N-linked glycosylation sites of the MCSP ectodomain. This is in contrast to the corresponding NG2 domain that features eight cysteines, one serine/glycine pair and five of its 11 potential N-linked glycosylation sites. The major difference between the deduced sequences of NG2 and MCSP is evident between amino acid residues 2043 and 2091. Thus, for NG2, it is reported that a cluster of six cysteine residues is present in this region (Nishiyama et al., supra, Fig. 3). An alignment of the MCSP and NG2 gene portion encoding for this region reveals three additional bases in the MCSP sequence, which are not found in the NG2 gene. The first additional base in position 6128 (SEQ ID NO: 1 ) causes a difference in the reading frame, which continues after the second additional base in position 6244, but is resolved after the third additional base in position 6273. The three additional bases are found both in the λgtl 1 (??) cDNA clone and in several independent PCR clones derived from the human melanoma cell lines M21 and A375-Met, respectively. Example 2: Localization of MCSP-Encoding mRNA in Melanoma Lesions by In Situ Hybridization Three cDNA fragments corresponding to different regions of the MCSP core protein coding sequence are used as riboprobe templates for in situ hybridization experiments. Clones 11, ra23 and an44 (cf. Table 1) carry MCSP core protein inserts of 559 bp (3756-4314 in pGEM-1), 811 pb (1431-2241 in pBluescript KS) and 677 bp (42113-4889 in pGEM-1), respectively. Sense and anti-sense RNA probes are labeled according to the instructions of the manufacturer (RNA Transkription Kit, Boehringer) with αS-^-UTP (more than 400 Ci/mmol, Amersham) to a specific activity of more than 10 9 dpm/μg. Labeled riboprobes are extracted with phenol chloroform and free nucleotides are removed by passage over a Sephadex G50 column. RNA is precipitated in 2.2 M ammonium acetate in 77% ethanol overnight at -20°C and is resuspended to an approximate activity of 250,000 cpm/μl (5x concentrated stock solution) in 50% v/v dionized formamide containing 20 mM dithiothreithol and stored at -70°C. Biopsies of melanoma skin metastases, primary melanomas and benign nevi are fixed in 4% paraformaldehyde in phosphate buffered salt solution (PBS) and then embedded in paraffin. Paraffin sections (8 μm) are placed on 3-aminoprophyltriethoxysilane-treated slides, which bind sections covalently to the glass surface and prevent loss of sections during experimental procedures. Paraffin sections are deparaffinized in xylene and absolute ethanol and air dried. Following rehydration with ethanol solutions of decreasing concentrations, sections are postfixed with 4% paraformaldehyde in PBS for 5 min, rinsed in PBS and water and depurinated for 20 min with 0.2 N HC1 at room temperature. These sections are then treated for 30 min with 2xSSC(0.3 M NaCl, 0.03 M Na-citrate, pH 7.0) at 70°C, dehydrated with increasing ethanol solutions and finally air dried. Pre-hybridization is performed at 54°C for 3 hrs in a solution of 50% v/v deionized formamide, 10% w/v dextransulfate, 0.3 M NaCl, 10 mM Tris, 10 mM sodium phosphate pH 6.8, 20 mM dithiothreitol, 0.2xDenhardts reagent 0.1 mg/ml Escherichia coli RNA and 0.5 μM non-radioactive αS-UTP. Hybridization is done overnight in the same solution, supplemented with 5xl0 4 cpm/μl αS^-UTP-labeled RNA probe in a humified chamber at 54°C. Slides are washed in the hybridization solution lacking dextransulfate, RNA and non-radioactive UTP, but containing 50% v/v deionized formamide and 10 mM dithiothreitol at 55°C, two times for 1 hr, and equilibrated for 15 min in a buffer solution consisting of 0.5 M NaCl, 10 mM Tris, 1 mM EDTA, 10 mM dithiothreitol, pH 7.5. Sections are then treated with 50 μg/ml RNase A in equilibration buffer for 30 min at 37°C to remove non-specifically bound probe. This is followed by washing in 2xSSC for 1 hr and then in O.lxSSC for 1 hr at 37°C. Slides are sequentially dehydrated in 65%, 85% and 95% (v/v) ethanol solutions containing 300 mM ammonium acetate and in absolute ethanol before being air dried. Sections are coated with a 1.2 dilution of Ilford K5 photoemulsion, air dried and exposed for 12 days in a light safe box containing silica gel at 4°C. The slides are then placed into D19 developer (Kodak), fixed in 30% sodium thiosulfate and stained with Haematoxylin and Eosin. The pattern of hybridization signals on autoradiographed sections is analyzed with a photomicroscope and brightfield/darkfield illuminations. All three probes of the MCSP coding sequence reveal comparable results. Biopsies from melanoma skin metastases that react strongly with MCSP-specific antibodies mAb 9.2.27 (Morgan et al., Hybridoma 1, 27-36 (1981)) or 763.74 (Giacomini et al., J. Immunol. 135, 696 (1985)) show abundant hybridization signals in cancer cells with all three anti-sense RNA probes and only background hybridization with the control sense RNA probes. Some hybridization is also detected in samples of benign nevi and normal epidermis, which exhibit no abundant staining with MCSP-specific antibodies. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: CIBA-GEIGY AG (B) STREET: Klybeckstr. 141 (C) CITY: Basel (E) COUNTRY: SCHWEIZ (F) POSTAL CODE (ZIP) : 4002 (G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991 (ii) TITLE OF INVENTION: Melanoma-associated Protein (iii) NUMBER OF SEQUENCES: 35 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7918 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..6966 (D) OTHER INFORMATION: /product= "MCSP" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATIO :1..87 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:88..6966 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CAG TCC GGC CGC GGC CCC CCA CTT CCA GCC CCC GGC CTG GCC TTG Met Gin Ser Gly Arg Gly Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu -29 -25 -20 -15 GCT TTG ACC CTG ACT ATG TTG GCC AGA CTT GCA TCC GCG GCT TCC TTC Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser- Phe -10 -5 1 TTC GGT GAG AAC CAC CTG GAG GTG CCT GTG GCC ACG GCT CTG ACC GAC Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp 5 10 15 ATA GAC CTG CAG CTG CAG TTC TCC ACG TCC CAG CCC GAA GCC CTC CTT lie Asp Leu Gin Leu Gin Phe Ser Thr Ser Gin Pro Glu Ala Leu Leu 20 25 30 35 CTC CTG GCA GCA GGC CCA GCT GAC CAC CTC CTG CTG CAG CTC TAC TCT Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gin Leu Tyr Ser 40 45 50 GGA CGC CTG CAG GTC AGA CTT GTT CTG GGC CAG GAG GAG CTG AGG CTG Gly Arg Leu Gin Val Arg Leu Val Leu Gly Gin Glu Glu Leu Arg Leu 55 60 65 CAG ACT CCA GCA GAG ACG CTG CTG AGT GAC TCC ATC CCC CAC ACT GTG Gin Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser lie Pro His Thr Val 70 75 80 GTG CTG ACT GTC GTA GAG GGC TGG GCC ACG TTG TCA GTC GAT GGG TTT Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe 85 90 95 CTG AAC GCC TCC TCA GCA GTC CCA GGA GCC CCC CTA GAG GTC CCC TAT Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr 100 105 110 115 GGG CTC TTT GTT GGG GGC ACT GGG ACC CTT GGC CTG CCC TAC CTG AGG Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg 120 125 130 GGA ACC AGC CGA CCC CTG AGG GGT TGC CTC CAT GCA GCC ACC CTC AAT Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn 135 140 145 GGC CGC AGC CTC CTC CGG CCT CTG ACC CCC GAT GTG CAT GAG GGC TGT Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys 150 155 160 GCT GAA GAG TTT TCT GCC AGT GAT GAT GTG GCC CTG GGC TTC TCT GGG Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly 165 170 175 CCC CAC TCT CTG GCT GCC TTC CCT GCC TGG GGC ACT CAG GAC GAA GGA Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gin Asp Glu Gly 180 185 190 195 ACC CTA GAG TTT ACA CTC ACC ACA CAG AGC CGG CAG GCA CCC TTG GCC Thr Leu Glu Phe Thr Leu Thr Thr Gin Ser Arg Gin Ala Pro Leu Ala 200 205 210 TTC CAG GCA GGG GGC CGG CGT GGG GAC TTC ATC TAT GTG GAC ATA TTT Phe Gin Ala Gly Gly Arg Arg Gly Asp Phe lie Tyr Val Asp lie Phe 215 220 225 GAG GGC CAC CTG CGG GCC GTG GTG GAG AAG GGC CAG GGT ACC GTA TTG Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gin Gly Thr Val Leu 230 235 240 CTC CAC AAC AGT GTG CCT GTG GCC GAT GGG CAG CCC CAT GAG GTC AGT Leu His Asn Ser Val Pro Val Ala Asp Gly Gin Pro His Glu Val Ser 245 250 255 GTC CAC ATC AAT GCT CAC CGG CTG GAA ATC TCC GTG GAC CAG TAC CCT Val His lie Asn Ala His Arg Leu Glu lie Ser Val Asp Gin Tyr Pro 260 265 270 275 ACG CAT ACT TCG AAC CGA GGA GTC CTC AGC TAC CTG GAG CCA CGG GGC Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg_ Gly 280 285 290 AGT CTC CTT CTC GGG GGG CTG GAT GCA GAG GCC TCT CGT CAC CTC CAG Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gin 295 300 305 GAA CAC CGC CTG GGC CTG ACA CCA GAG GCC ACC AAT GCC TCC CTG CTG Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu 310 315 320 GGC TGC ATG GAA GAC CTC AGT GTC AAT GGC CAG AGG CGG GGG CTG CGG Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gin Arg Arg Gly Leu Arg 325 330 335 GAA GCT TTG CTG ACG CGC AAC ATG GCA GCC GGC TGC AGG CTG GAG GAG Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu 340 345 350 355 GAG GAG TAT GAG GAC GAT GCC TAT GGC CAT TAT GAA GCT TTC TCC ACC Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr 360 365 370 CTG GCT CCC GAG GCT TGG CCA GCC ATG GAG CTG CCT GAG CCA TGC GTG Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val 375 380 385 CCT GAG CCA GGG CTG CCT CCT GTC TTT GCC AAT TTC ACC CAG CTG CTG Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gin Leu Leu 390 395 400 ACT ATC AGC CCA CTG GTG GTG GCC GAG GGT GGC ACA GCC TGG CTT GAG Thr lie Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu 405 410 415 TGG AGG CAT GTG CAG CCC ACG CTG GAC CTG ATG GAG GCT GAG CTG CGC Trp Arg His Val Gin Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg 420 425 430 435 AAA TCC CAG GTG CTG TTC AGC GTG ACC CGA GGG GCA CAC TAT GGC GAG Lys Ser Gin Val Leu Phe Ser Val Thr Arg Gly Ala His Tyr Gly Glu 440 445 450 CTC GAG CTG GAC ATC CTG GGT GCC CAG GCA CGA AAA ATG TTC ACC CTC Leu Glu Leu Asp lie Leu Gly Ala Gin Ala Arg Lys Met Phe Thr Leu 455 460 465 CTG GAC GTG GTG AAC CGC AAG GCC CGC TTC ATC CAC GAT GGC TCT GAG Leu Asp Val Val Asn Arg Lys Ala Arg Phe lie His Asp Gly Ser Glu 470 475 480 GAC ACC TCC GAC CAG CTG GTG CTG GAG GTG TCG GTG ACG GCT CGG GTG Asp Thr Ser Asp Gin Leu Val Leu Glu Val Ser Val Thr Ala Arg Val 485 490 495 CCC ATG CCC TCA TGC CTT CGG AGG GGC CAA ACA TAC CTC CTG CCC ATC Pro Met Pro Ser Cys Leu Arg Arg Gly Gin Thr Tyr Leu Leu Pro lie 500 505 510 515 CAG GTC AAC CCT GTC AAT GAC CCA CCC CAC ATC ATC TTC CCA CAT GGC Gin Val Asn Pro Val Asn Asp Pro Pro His lie lie Phe Pro His Gly 520 525 530 AGC CTC ATG GTG ATC CTG GAA CAC ACG CAG AAG CCG CTG GGG CCT GAG Ser Leu Met Val lie Leu Glu His Thr Gin Lys Pro Leu Gly Pro Glu 535 540 545 GTT TTC CAG GCC TAT GAC CCG GAC TCT GCC TGT GAG GGC CTC ACC TTC Val Phe Gin Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe 550 555 560 CAG GTC CTT GGC ACC TCC TCT GGC CTC CCC GTG GAG CGC CGA GAC CAG Gin Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp- Gin 565 570 575 CCT GGG GAG CCG GCG ACC GAG TTC TCC TGC CGG GAG TTG GAG GCC GGC Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly 580 585 590 595 AGC CTA GTC TAT GTC CAC TGC GGT GGT CCT GCA CAG GAC TTG ACG TTC Ser Leu Val Tyr Val His Cys Gly Gly Pro Ala Gin Asp Leu Thr Phe 600 605 610 CGG GTC AGC GAT GGA CTG CAG GCC AGC CCC CCG GCC ACG CTG AAG GTG Arg Val Ser Asp Gly Leu Gin Ala Ser Pro Pro Ala Thr Leu Lys Val 615 620 625 GTG GCC ATC CGG CCG GCC ATA CAG ATC CAC CGC AGC ACA GGG TTG CGA Val Ala lie Arg Pro Ala lie Gin lie His Arg Ser Thr Gly Leu Arg 630 635 640 CTG GCC CAA GGC TCT GCC ATG CCC ATC TTG CCC GCC AAC CTG TCG GTG Leu Ala Gin Gly Ser Ala Met Pro lie Leu Pro Ala Asn Leu Ser Val 645 650 655 GAG ACC AAT GCC GTG GGG CAG GAT GTG AGC GTG CTG TTC CGC GTC ACT Glu Thr Asn Ala Val Gly Gin Asp Val Ser Val Leu Phe Arg Val Thr 660 665 670 675 GGG GCC CTG CAG TTT GGG GAG CTG CAG AAG CAT AGT ACA GGT GGG GTG Gly Ala Leu Gin Phe Gly Glu Leu Gin Lys His Ser Thr Gly Gly Val 680 685 690 GAG GGT GCT GAG TGG TGG GCC ACA CAG GCG TTC CAC CAG CGG GAT GTG Glu Gly Ala Glu Trp Trp Ala Thr Gin Ala Phe His Gin Arg Asp Val 695 700 705 GAG CAG GGC CGC GTG AGG TAC CTG AGC ACT GAC CCA CAG CAC CAC GCT Glu Gin Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gin His His .Ala 710 715 720 TAC GAC ACC GTG GAG AAC CTG GCC CTG GAG GTG CAG GTG GGC CAG GAG Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gin Val Gly Gin Glu 725 730 735 ATC CTG AGC AAT CTG TCC TTC CCA GTG ACC ATC CAG AGA GCC ACT GTG lie Leu Ser Asn Leu Ser Phe Pro Val Thr lie Gin Arg Ala Thr Val 740 745 750 755 TGG ATG CTG CGG CTG GAG CCA CTG CAC ACT CAG AAC ACC CAG CAG GAG Trp Met Leu Arg Leu Glu Pro Leu His Thr Gin Asn Thr Gin Gin Glu 760 765 770 ACC CTC ACC ACA GCC CAC CTG GAG GCC ACC CTG GAG GAG GCA GGC CCA Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro 775 780 785 AGC CCC CCA ACC TTC CAT TAT GAG GTG GTT CAG GCT CCC AGG AAA GGC Ser Pro Pro Thr Phe His Tyr Glu Val Val Gin Ala Pro Arg Lys Gly 790 795 800 AAC CTT CAA CTA CAG GGC ACA AGG CTG TCA GAT GGC CAG GGC TTC ACC Asn Leu Gin Leu Gin Gly Thr Arg Leu Ser Asp Gly Gin Gly Phe Thr 805 810 815 CAG GAT GAC ATA CAG GCT GGC CGG GTG ACC TAT GGG GCC ACA GCT CGT Gin Asp Asp lie Gin Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg 820 825 830 835 GCC TCA GAG GCA GTC GAG GAC ACC TTC CGT TTC CGT GTC ACA GCT CCA Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro 840 845 850 CCA TAT TTC TCC CCA CTC TAT ACC TTC CCC ATC CAC ATT GGT GGT GAC Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro lie His lie Gly Gly-Asp 855 860 865 CCA GAT GCG CCT GTC CTC ACC AAT GTC CTC CTC GTG GTG CCT GAG GGT Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly 870 875 880 GGT GAG GGT GTC CTC TCT GCT GAC CAC CTC TTT GTC AAG AGT CTC AAC Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn 885 890 895 AGT GCC AGC TAC CTC TAT GAG GTC ATG GAG CGG CCC CGC CTT GGG AGG Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg Leu Gly Arg 900 905 910 915 TTG GCT TGG CGT GGG ACA CAG GAC AAG ACC ACT ATG GTG ACA TCC TTC Leu Ala Trp Arg Gly Thr Gin Asp Lys Thr Thr Met Val Thr Ser Phe 920 925 930 ACC AAT GAA GAC CTG TTG CGT GGC CGG CTG GTC TAC CAG CAT GAT GAC Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gin His Asp Asp 935 940 945 TCC GAG ACC ACA GAA GAT GAT ATC CCA TTT GTT GCT ACC CGC CAG GGC Ser Glu Thr Thr Glu Asp Asp He Pro Phe Val Ala Thr Arg Gin Gly 950 955 960 GAG AGC AGT GGT GAC ATG GCC TGG GAG GAG GTA CGG GGT GTC TTC CGA Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg 965 970 975 GTG GCC ATC CAG CCC GTG AAT GAC CAC GCC CCT GTG CAG ACC ATC AGC Val Ala He Gin Pro Val Asn Asp His Ala Pro Val Gin Thr He Ser 980 985 990 995 CGG ATC TTC CAT GTG GCC CGG GGT GGG CGG CGG CTG CTG ACT ACA GAC Arg He Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr Thr -Asp 1000 1005 1010 GAC GTG GCC TTC AGC GAT GCT GAC TCG GGC TTT GCT GAC GCC GAG CTG Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp Ala Gin Leu 1015 1020 1025 GTG CTT ACC CGC AAG GAC CTC CTC TTT GGC AGT ATC GTG GCC GTA GAT Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser He Val Ala Val Asp 1030 1035 1040 GAG CCC ACG CGG CCC ATC TAC CGC TTC ACC CAG GAG GAC CTC AGG AAG Glu Pro Thr Arg Pro He Tyr Arg Phe Thr Gin Glu Asp Leu Arg Lys 1045 1050 1055 AGG CGA GTA CTG TTC GTG CAC TCA GGG GCT GAC CGT GGC TGG ATC CAG Arg Arg Val Leu Phe Val His Ser Gly Ala Asp Arg Gly Trp He Gin 1060 1065 1070 1075 CTG CAG GTG TCC GAC GGG CAA CAC CAG GCC ACT GCG CTG CTG GAG GTG Leu Gin Val Ser Asp Gly Gin His Gin Ala Thr Ala Leu Leu Glu Val 1080 1085 1090 CAG GCC TCG GAA CCC TAC CTC CGT GTG GCC AAC GGC TCC AGC CTT GTG Gin Ala Ser Glu Pro Tyr Leu Arg Val Ala Asn Gly Ser Ser Leu Val 1095 1100 1105 GTC CCT CAA GGA GGC CAG GGC ACC ATC GAC ACG GCC GTG CTC CAC CTG Val Pro Gin Gly Gly Gin Gly Thr He Asp Thr Ala Val Leu His Leu 1110 1115 1120 GAC ACC AAC CTC GAC ATC CGC AGT GGG GAT GAG GTC CAC TAC CAC GTC Asp Thr Asn Leu Asp He Arg Ser Gly Asp Glu Val His Tyr His Val 1125 1130 1135 ACA GCT GGC CCT CGC TGG GGA CAA CTA GTC CGG GCT GGT CAG CCA GCC Thr Ala Gly Pro Arg Trp Gly Gin Leu Val Arg Ala Gly Gin Pro -Ala 1140 1145 1150 1155 ACA GCC TTC TCC CAG CAG GAC CTG CTG GAT GGG GCC GTT CTC TAT AGC Thr Ala Phe Ser Gin Gin Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser 1160 1165 1170 CAC AAT GGC AGC CTC AGC CCC GAA GAC ACC ATG GCC TTC TCC GTG GAA His Asn Gly Ser Leu Ser Pro Glu Asp Thr Met Ala Phe Ser Val Glu 1175 1180 1185 GCA GGG CCA GTG CAC ACG GAT GCC ACC CTA CAA GTG ACC ATT GCC CTA Ala Gly Pro Val His Thr Asp Ala Thr Leu Gin Val Thr He Ala Leu 1190 1195 1200 GAG GGC CCA CTG GCC CCA CTG AAG CTG GTC CGG CAC AAG AAG ATC TAC Glu Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys He Tyr 1205 1210 1215 GTC TTC CAG GGA GAG GCA GCT GAG ATC AGA AGG GAC CAG CTG GAG GCA Val Phe Gin Gly Glu Ala Ala Glu He Arg Arg Asp Gin Leu Glu Ala 1220 1225 1230 1235 GCC CAG GAG GCA GTG CCA CCT GCA GAC ATC GTA TTC TCA GTG AAG AGC Ala Gin Glu Ala Val Pro Pro Ala Asp He Val Phe Ser Val Lys Ser 1240 1245 1250 CCA CCG AGT GCC GGC TAC CTG GTG ATG GTG TCG CGT GGC GCC TTG GCA Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala 1255 1260 1265 GAT GAG CCA CCC AGC CTG GAC CCT GTG CAG AGC TTC TCC CAG GAG GCA Asp Glu Pro Pro Ser Leu Asp Pro Val Gin Ser Phe Ser Gin Glu Ala 1270 1275 1280 GTG GAC ACA GGC AGG GTC CTG TAC CTG CAC TCC CGC CCT GAG GCC TGG Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser Arg Pro Glu Ala Trp 1285 1290 1295 AGC GAT GCC TTC TCG CTG GAT GTG GCC TCA GGC CTG GGT GCT CCC CTC Ser Asp Ala Phe Ser Leu Asp Val Ala Ser Gly Leu Gly Ala Pro Leu 1300 1305 1310 1315 GAG GGC GTC CTT GTG GAG CTG GAG GTG CTG CCC GCT GCC ATC CCA CTA Glu Gly Val Leu Val Glu Leu Glu Val Leu Pro Ala Ala He Pro Leu 1320 1325 1330 GAG GCG CAA AAC TTC AGC GTC CCT GAG GGT GGC AGC CTC ACC CTG GCC Glu Ala Gin Asn Phe Ser Val Pro Glu Gly Gly Ser Leu Thr Leu Ala 1335 1340 1345 CCT CCA CTG CTC CGT GTC TCC GGG CCC TAC TTC CCC ACT CTC CTG GGC Pro Pro Leu Leu Arg Val Ser Gly Pro Tyr Phe Pro Thr Leu Leu Gly 1350 1355 1360 CTC AGC CTG CAG GTG CTG GAG CCA CCC CAG CAT GGA CCC CTG CAG AAG Leu Ser Leu Gin Val Leu Glu Pro Pro Gin His Gly Pro Leu Gin Lys 1365 1370 1375 GAG GAC GGA CCT CAA GCC AGG ACC CTC AGC GCC TTC TCC TGG AGA ATG Glu Asp Gly Pro Gin Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met 1380 1385 1390 1395 GTG GAA GAG CAG CTG ATC CGC TAC GTG CAT GAC GGG AGC GAG ACA CTG Val Glu Glu Gin Leu He Arg Tyr Val His Asp Gly Ser Glu Thr Leu 1400 1405 1410 ACA GAC AGT TTT GTC CTG ATG GCT AAT GCC TCC GAG ATG GAT CGC CAG Thr Asp Ser Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gin 1415 1420 1425 AGC CAT CCT GTG GCC TTC ACT GTC ACT GTC CTG CCT GTC AAT GAC CAA Ser His Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp " Gin 1430 1435 1440 CCC CCC ATC CTC ACT ACA AAC ACA GGC CTG CAG ATG TGG GAG GGG GCC Pro Pro He Leu Thr Thr Asn Thr Gly Leu Gin Met Trp Glu Gly Ala 1445 1450 1455 ACT GCG CCC ATC CCT GCG GAG GCT CTG AGG AGC ACG GAC GGC GAC TCT Thr Ala Pro He Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp Ser 1460 1465 1470 1475 GGG TCT GAG GAT CTG GTC TAC ACC ATC GAG CAG CCC AGC AAC GGG CGG Gly Ser Glu Asp Leu Val Tyr Thr He Glu Gin Pro Ser Asn Gly Arg 1480 1485 1490 GTA GTG CTG CGG GGG GCG CCG GGC ACT GAG GTG CGC AGC TTC ACG CAG Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser Phe Thr Gin 1495 1500 1505 GCC CAG CTG GAC GGC GGG CTC GTG CTG TTC TCA CAC AGA GGA ACC CTG Ala Gin Leu Asp Gly Gly Leu Val Leu Phe Ser His Arg Gly Thr Leu 1510 1515 1520 GAT GGA GGC TTC CCG TTC CGC CTC TCT GAC GGC GAG CAC ACT TCC CCC Asp Gly Gly Phe Pro Phe Arg Leu Ser Asp Gly Glu His Thr Ser Pro 1525 1530 1535 GGA CAC TTC TTC CGA GTG ACG GCC CAG AAG CAA GTG CTC CTC TCG CTG Gly His Phe Phe Arg Val Thr Ala Gin Lys Gin Val Leu Leu Ser Leu 1540 1545 1550 1555 AAG GGC AGC CAG ACA CTG ACT GTC TGC CCA GGG TCC GTC CAG CCA CTC Lys Gly Ser Gin Thr Leu Thr Val Cys Pro Gly Ser Val Gin Pro Leu 1560 1565 1570 AGC AGT CAG ACC CTC AGG GCC AGC TCC AGC GCA GGC ACT GAC CCC CAG Ser Ser Gin Thr Leu Arg Ala Ser Ser Ser Ala Gly Thr Asp Pro Gin 1575 1580 1585 CTC CTG CTC TAC CGT GTG GTG CGG GGC CCC CAG CTA GGC CGG CTG TTC Leu Leu Leu Tyr Arg Val Val Arg Gly Pro Gin Leu Gly Arg Leu Phe 1590 1595 1600 CAC GCC CAG CAG GAC AGC ACA GGG GAG GCC CTG GTG AAC TTC ACT CAG His Ala Gin Gin Asp Ser Thr Gly Glu Ala Leu Val Asn Phe Thr Gin 1605 1610 1615 GCA GAG GTC TAC GCT GGG AAT ATT CTG TAT GAG CAT GAG ATG CCC CCC Ala Glu Val Tyr Ala Gly Asn He Leu Tyr Glu His Glu Met Pro Pro 1620 1625 1630 1635 GAG CCC TTT TGG GAG GCC CAT GAT ACC CTA GAG CTC CAG CTG TCC TCG Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gin Leu Ser Ser 1640 1645 1650 CCG CCT GCC CGG GAC GTG GCC GCC ACC CTT GCT GTG GCT GTG TCT TTT Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe 1655 1660 1665 GAG GCT GCC TGT CCC CAG CGC CCC AGC CAC CTC TGG AAG AAC AAA GGT Glu Ala Ala Cys Pro Gin Arg Pro Ser His Leu Trp Lys Asn Lys Gly 1670 1675 1680 CTC TGG GTC CCC GAG GGC CAG CGG GCC AGG ATC ACC GTG GCT GCT CTG Leu Trp Val Pro Glu Gly Gin Arg Ala Arg He Thr Val Ala Ala Leu 1685 1690 1695 GAT GCC TCC AAT CTC TTG GCC AGC GTT CCA TCA CCC CAG CGC TCA GAG Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gin Arg Ser Glu 1700 1705 1710 1715 CAT GAT GTG CTC TTC CAG GTC ACA CAG TTC CCC AGC CGG GGC CAG CTG His Asp Val Leu Phe Gin Val Thr Gin Phe Pro Ser Arg Gly Gin Leu 1720 1725 1730 TTG GTG TCC GAG GAG CCC CTC CAT GCT GGG CAG CCC CAC TTC CTG CAG Leu Val Ser Glu Glu Pro Leu His Ala Gly Gin Pro His Phe Leu Gin 1735 1740 1745 TCC CAG CTG GCT GCA GGG CAG CTA GTG TAT GCC CAC GGC GGT GGG GGC Ser Gin Leu Ala Ala Gly Gin Leu Val Tyr Ala His Gly Gly Gly Gly 1750 1755 1760 ACC CAG CAG GAT GGC TTC CAC TTT CGT GCC CAC CTC CAG GGG CCA GCA Thr Gin Gin Asp Gly Phe His Phe Arg Ala His Leu Gin Gly Pro Ala 1765 1770 1775 GGG GCC TCC GTG GCT GGA CCC CAA ACC TCA GAG GCC TTT GCC ATC ACG Gly Ala Ser Val Ala Gly Pro Gin Thr Ser Glu Ala Phe Ala He Thr 1780 1785 1790 1795 GTG AGG GAT GTA AAT GAG CGG CCC CCT CAG CCA CAG GCC TCT GTC CCA Val Arg Asp Val Asn Glu Arg Pro Pro Gin Pro Gin Ala Ser Val Pro 1800 1805 1810 CTC CGG CTC ACC CGA GGC TCT CGT GCC CCC ATC TCC CGG GCC CAG CTG Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro He Ser Arg Ala Gin Leu 1815 1820 1825 AGT GTG GTG GAC CCA GAC TCA GCT CCT GGG GAG ATT GAG TAC GAG GTC Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu He Glu Tyr Glu Val 1830 1835 1840 CAG CGG GCA CCC CAC AAC GGC TTC CTC AGC CTG GTG GGT GGT GGC CTG Gin Arg Ala Pro His Asn Gly Phe Leu Ser Leu Val Gly Gly Gly Leu 1845 1850 1855 GGG CCC GTG ACC CGC TTC ACG CAA GCC GAT GTG GAT TCA GGG CGG CTG Gly Pro Val Thr Arg Phe Thr Gin Ala Asp Val Asp Ser Gly Arg Leu 1860 1865 1870 1875 GCC TTC GTG GCC AAC GGG AGC AGC GTG GCA GGC ATC TTC CAG CTG AGC Ala Phe Val Ala Asn Gly Ser Ser Val Ala Gly He Phe Gin Leu Ser 1880 1885 1890 ATG TCT GAT GGG GCC AGC CCA CCC CTG CCC ATG TCC CTG GCT GTG GAC Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp 1895 1900 1905 ATC CTA CCA TCC GCC ATC GAG GTG CAG CTG CGG GCA CCC CTG GAG GTG He Leu Pro Ser Ala He Glu Val Gin Leu Arg Ala Pro Leu Glu Val 1910 1915 1920 CCC CAA GCT TTG GGG CGC TCC TCA CTG AGC CAG CAG CAG CTC CGG GTG Pro Gin Ala Leu Gly Arg Ser Ser Leu Ser Gin Gin Gin Leu Arg Val 1925 1930 1935 GTT TCA GAT CGG GAG GAG CCA GAG GCA GCA TAC CGC CTC ATC CAG GGA Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu He Gin Gly 1940 1945 1950 1955 CCC CAG TAT GGG CAT CTC CTG GTG GGC GGG CGG CCC ACC TCG GCC TTC Pro Gin Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser Ala Phe 1960 1965 1970 AGC CAA TTC CAG ATA GAC CAG GGC GAG GTG GTC TTT GCC TTC ACC AAC Ser Gin Phe Gin He Asp Gin Gly Glu Val Val Phe Ala Phe Thr Asn 1975 1980 1985 TTC TCC TCC TCT CAT GAC CAC TTC AGA GTC CTG GCA CTG GCT AGG GGT Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala Leu Ala Arg Gly 1990 1995 2000 GTC AAT GCA TCA GCC GTA GTG AAC GTC ACT GTG AGG GCT CTG CTG CAT Val Asn Ala Ser Ala Val Val Asn Val Thr Val Arg Ala Leu Leu- His 2005 2010 2015 GTG TGG GCA GGT GGG CCA TGG CCC CAG GGT GCC ACC CTG CGC CTG GAC Val Trp Ala Gly Gly Pro Trp Pro Gin Gly Ala Thr Leu Arg Leu Asp 2020 2025 2030 2035 CCC ACC GTC CTA GAT GCT GGC GAG CTG GCC AAC CGC ACA GGC AGT GTG Pro Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr Gly Ser Val 2040 2045 2050 CCG CGC TTC CGC CTC CTG GAG GGA CCC CGG CAT GGC CGC GTG GTC CGC Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His Gly Arg Val Val Arg 2055 2060 2065 GTG CCC CGA GCC AGG ACG GAG CCC GGG GGC AGC CAG CTG GTG GAG CAG Val Pro Arg Ala Arg Thr Glu Pro Gly Gly Ser Gin Leu Val Glu Gin 2070 2075 2080 TTC ACT CAG CAG GAC CTT GAG GAC GGG AGG CTG GGG CTG GAG GTG GGC Phe Thr Gin Gin Asp Leu Glu Asp Gly Arg Leu Gly Leu Glu Val Gly 2085 2090 2095 AGG CCA GAG GGG AGG GCC CCC GGC CCC GCA GGT GAC AGT CTC ACT CTG Arg Pro Glu Gly Arg Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu 2100 2105 2110 2115 GAG CTG TGG GCA CAG GGC GTC CCG CCT GCT GTG GCC ' TCC CTG GAC TTT Glu Leu Trp Ala Gin Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe 2120 2125 2130 GCC ACT GAG CCT TAC AAT GCT GCC CGG CCC TAC AGC GTG GCC CTG CTC Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu 2135 2140 2145 AGT GTC CCC GAG GCC GCC CGG ACG GAA GCA GGG AAG CCA GAG AGC AGC Ser Val Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser 2150 2155 2160 ACC CCC ACA GGC GAG CCA GGC CCC ATG GCA TCC AGC CCT GAG CCC GCT Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala 2165 2170 2175 GTG GCC AAG GGA GGC TTC CTG AGC TTC CTT GAG GCC AAC ATG TTC AGC Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe Ser 2180 2185 2190 2195 GTC ATC ATC CCC ATG TGC CTG GTA CTT CTG CTC CTG GCG CTC ATC CTG Val He He Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu He Leu 2200 2205 2210 CCC CTG CTC TTC TAC CTC CGA AAA CGC AAC AAG ACG GGC AAG CAT GAC Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly Lys His Asp 2215 2220 2225 GTC CAG GTC CTG ACT GCC AAG CCC CGC AAC GGC CTG GCT GGT GAC ACC Val Gin Val Leu Thr Ala Lys Pro Arg Asn Gly Leu Ala Gly Asp Thr 2230 2235 2240 GAG ACC TTT CGC AAG GTG GAG CCA GGC CAG GCC ATC CCG CTC ACA GCT Glu Thr Phe Arg Lys Val Glu Pro Gly Gin Ala He Pro Leu Thr Ala 2245 2250 2255 GTG CCT GGC CAG GGG CCC CCT CCA GGA GGC CAG CCT GAC CCA GAG CTG Val Pro Gly Gin Gly Pro Pro Pro Gly Gly Gin Pro Asp Pro Glu Leu 2260 2265 2270 2275 CTG CAG TTC TGC CGG ACA CCC AAC CCT GCC CTT AAG AAT GGC CAG TAC Leu Gin Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly Gin Tyr 2280 2285 2290 TGG GTG TGAAGGCCTG GCCTGGGCCC AGATGCTGAT CGGGCCAGGG ACAGGCTTGC Trp Val CCATGTCCCG GGCCCCATTG CTTCCATGCC CGGTGCTGTC TGAGTATCCC CAGAGCAAGA 707 GAGACCTGGA GACACCAGGG GTGGAGGGTC CTGGGAGATA GTCCCAGGGG TCCGGGACAG 713 AGTGGAGTCA AGAGCTGGAA CCTCCCTCAG CTCACTCCGA GCCTGGAGAA CTGCAGGGGC 719 CAAGGTGGAG GCAGGCTTAA GTTCAGTCCT CCTGCCCTGG AGCTGGTTTG GGCTGTCAAA 725 ACCAGGGTAA CCTCCTACAT GGGTCATGAC TCTGGG CCT GGGTCTGTGA CCTTGGGTAA 73] GTCGCGCCTG ACCCAGGCTG CTAAGAGGGC AAGGAGAAGG AAGTACCCTG GGGAGGGAAG 737 GGACAGAGGA AGCTATTCCT GGCTTTTCTA CTCCAACCCA GGCCACCCTT TGTCTCTNCC 741 CCAGAGTTGA GAAAAAAACT TCCTCCCCTG GTTTTTTAGG GAG TGGTAT CCCCTGGAGT 749 AGAGGGCAAG AGGAGAGAGC GCCTCCAGTC TAGAAGGCAT AAGCCAATAG GATAATATAT 755 TCAGGGTGCA GGGTGGGTAG ATTTAAGGGA GATTGCAAGG 76 AAGCTATTTA ACATGGTGCT GAGCTAGCCA GGACTGATGG AGCCCCTGGG GGTGTGGGAT 767 GGAGGAGGGT CTGCAGCCAG TTCATTCCCA GGGCCCCATC TTGATGGGCC AAGGGCTAAA 773 CATGCATGTG TCAGTGGCTT TGGAGCAGGC TAGGCTGGGG CTCATCGAGG GTCTCAGGCC 77 GAGGCCACTG TAGTGCC GT GCCCCCCTG GGACTAGGGC AGGCAGCTGG GGGCACTTGG 78^ TTCCATGGAG CCTGGATAAA CAGTGCTTTG GAGGCTCTGG AAAAAAAAAA AA.^.AAAAAA 79] AA 79] (2) INFORMATION FOR SEQ ID NO: 2: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2322 amino acids (B) TYPE: ammo ac d (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Gin Ser Gly Arg Gly Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu -29 -25 -20 -15 Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser Phe -10 -5 1 Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp 5 10 15 He Asp Leu Gin Leu Gin Phe Ser Thr Ser Gin Pro Glu Ala Leu Leu 20 25 30 35 Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gin Leu Tyr Ser 40 45 50 Gly Arg Leu Gin Val Arg Leu Val Leu Gly Gin Glu Glu Leu Arg Leu 55 60 65 Gin Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser He Pro His Thr Val 70 75 80 Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe 85 90 95 Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr 100 105 110 115 Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg 120 125 130 Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn 135 140 145 Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys 150 155 160 Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly 165 170 175 Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gin Asp Glu Gly 180 185 190 195 Thr Leu Glu Phe Thr Leu Thr Thr Gin Ser Arg Gin Ala Pro Leu Ala 200 205 210 Phe Gin Ala Gly Gly Arg Arg Gly Asp Phe He Tyr Val Asp He Phe 215 220 225 Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gin Gly Thr Val Leu 230 235 240 Leu His Asn Ser Val Pro Val Ala Asp Gly Gin Pro His Glu Val Ser 245 250 255 Val His He Asn Ala His Arg Leu Glu He Ser Val Asp Gin Tyr Pro 260 265 270 275 Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly 280 285 290 Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gin 295 300 305 Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu 310 315 320 Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gin Arg Arg Gly Leu Arg 325 330 335 Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu 340 345 350 355 Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr 360 365 370 Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val 375 380 385 Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gin Leu Leu 390 395 400 Thr He Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu 405 410 415 Trp Arg His Val Gin Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg 420 425 430 435 Lys Ser Gin Val Leu Phe Ser Val Thr Arg Gly Ala His Tyr Gly Glu 440 445 450 Leu Glu Leu Asp He Leu Gly Ala Gin Ala Arg Lys Met Phe Thr Leu 455 460 465 Leu Asp Val Val Asn Arg Lys Ala Arg Phe He His Asp Gly Ser Glu 470 475 480 Asp Thr Ser Asp Gin Leu Val Leu Glu Val Ser Val Thr Ala Arg Val 485 490 495 Pro Met Pro Ser Cys Leu Arg Arg Gly Gin Thr Tyr Leu Leu Pro He 500 505 510 515 Gin Val Asn Pro Val Asn Asp Pro Pro His He He Phe Pro His Gly 520 525 530 Ser Leu Met Val He Leu Glu His Thr Gin Lys Pro Leu Gly Pro Glu 535 540 545 Val Phe Gin Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe 550 555 560 Gin Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp Gin 565 570 575 Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly 580 585 590 595 Ser Leu Val Tyr Val His Cys Gly Gly Pro Ala Gin Asp Leu Thr Phe 600 605 610 Arg Val Ser Asp Gly Leu Gin Ala Ser Pro Pro Ala Thr Leu Lys Val 615 620 625 Val Ala He Arg Pro Ala He Gin He His Arg Ser Thr Gly Leu Arg 630 635 640 Leu Ala Gin Gly Ser Ala Met Pro He Leu Pro Ala Asn Leu Ser Val 645 650 655 Glu Thr Asn Ala Val Gly Gin Asp Val Ser Val Leu Phe Arg Val Thr 660 665 670 675 Gly Ala Leu Gin Phe Gly Glu Leu Gin Lys His Ser Thr Gly Gly Val 680 685 690 Glu Gly Ala Glu Trp Trp Ala Thr Gin Ala Phe His Gin Arg Asp Val 695 700 705 Glu Gin Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gin His His Ala 710 715 720 Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gin Val Gly Gin Glu 725 730 735 Ile Leu Ser Asn Leu Ser Phe Pro Val Thr He Gin Arg Ala Thr Val 740 745 750 755 Trp Met Leu Arg Leu Glu Pro Leu His Thr Gin Asn Thr Gin Gin Glu 760 765 770 Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro 775 780 785 Ser Pro Pro Thr Phe His Tyr Glu Val Val Gin Ala Pro Arg Lys Gly 790 795 800 Asn Leu Gin Leu Gin Gly Thr Arg Leu Ser Asp Gly Gin Gly Phe Thr 805 810 815 Gin Asp Asp He Gin Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg 820 825 830 835 Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro 840 845 850 Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro He His He Gly Gly Asp 855 860 865 Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Cly 870 875 880 Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn 885 890 895 Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg Leu Gly Arg 900 905 910 915 Leu Ala Trp Arg Gly Thr Gin Asp Lys Thr Thr Met Val Thr Ser Phe 920 925 930 Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gin His Asp Asp 935 940 945 Ser Glu Thr Thr Glu Asp Asp He Pro Phe Val Ala Thr Arg Gin Gly 950 955 960 Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg 965 970 975 Val Ala He Gin Pro Val Asn Asp His Ala Pro Val Gin Thr He Ser 980 985 990 995 Arg He Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr Thr Asp 1000 1005 1010 Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp Ala Gin Leu 1015 1020 1025 Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser He Val Ala Val Asp 1030 1035 1040 Glu Pro Thr Arg Pro He Tyr Arg Phe Thr Gin Glu Asp Leu Arg Lys 1045 1050 1055 Arg Arg Val Leu Phe Val His Ser Gly Ala Asp Arg Gly Trp He tSln 1060 1065 1070 1075 Leu Gin Val Ser Asp Gly Gin His Gin Ala Thr Ala Leu Leu Glu Val 1080 1085 1090 Gin Ala Ser Glu Pro Tyr Leu Arg Val Ala Asn Gly Ser Ser Leu Val 1095 1100 1105 Val Pro Gin Gly Gly Gin Gly Thr He Asp Thr Ala Val Leu His Leu 1110 1115 1120 Asp Thr Asn Leu Asp He Arg Ser Gly Asp Glu Val His Tyr His Val 1125 1130 1135 Thr Ala Gly Pro Arg Trp Gly Gin Leu Val Arg Ala Gly Gin Pro Ala 1140 1145 1150 1155 Thr Ala Phe Ser Gin Gin Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser 1160 1165 1170 His Asn Gly Ser Leu Ser Pro Glu Asp Thr Met Ala Phe Ser Val Glu 1175 1180 1185 Ala Gly Pro Val His Thr Asp Ala Thr Leu Gin Val Thr He Ala Leu 1190 1195 1200 Glu Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys He Tyr 1205 1210 1215 Val Phe Gin Gly Glu Ala Ala Glu He Arg Arg Asp Gin Leu Glu Ala 1220 1225 1230 1235 Ala Gin Glu Ala Val Pro Pro Ala Asp He Val Phe Ser Val Lys Ser 1240 1245 1250 Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala 1255 1260 1265 Asp Glu Pro Pro Ser Leu Asp Pro Val Gin Ser Phe Ser Gin Glu Ala 1270 1275 1280 Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser Arg Pro Glu Ala Trp 1285 1290 1295 Ser Asp Ala Phe Ser Leu Asp Val Ala Ser Gly Leu Gly Ala Pro Leu 1300 1305 1310 1315 Glu Gly Val Leu Val Glu Leu Glu Val Leu Pro Ala Ala He Pro Leu 1320 1325 1330 Glu Ala Gin Asn Phe Ser Val Pro Glu Gly Gly Ser Leu Thr Leu Ala 1335 1340 1345 Pro Pro Leu Leu Arg Val Ser Gly Pro Tyr Phe Pro Thr Leu Leu Gly 1350 1355 1360 Leu Ser Leu Gin Val Leu Glu Pro Pro Gin His Gly Pro Leu Gin Lys 1365 1370 1375 Glu Asp Gly Pro Gin Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met 1380 1385 1390 1395 Val Glu Glu Gin Leu He Arg Tyr Val His Asp Gly Ser Glu Thr Leu 1400 1405 1410 Thr Asp Ser Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gin 1415 1420 1425 Ser His Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp Gin 1430 1435 1440 Pro Pro He Leu Thr Thr Asn Thr Gly Leu Gin Met Trp Glu Gly Ala 1445 1450 1455 Thr Ala Pro He Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp Ser 1460 1465 1470 1475 Gly Ser Glu Asp Leu Val Tyr Thr He Glu Gin Pro Ser Asn Gly Arg 1480 1485 1490 Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser Phe Thr Gin 1495 1500 1505 - 57 - Ala Gin Leu Asp Gly Gly Leu Val Leu Phe Ser His Arg Gly Thr Leu 1510 1515 1520 Asp Gly Gly Phe Pro Phe Arg Leu Ser Asp Gly Glu His Thr Ser Pro 1525 1530 1535 Gly His Phe Phe Arg Val Thr Ala Gin Lys Gin Val Leu Leu Ser Leu 1540 1545 1550 1555 Lys Gly Ser Gin Thr Leu Thr Val Cys Pro Gly Ser Val Gin Pro Leu 1560 1565 1570 Ser Ser Gin Thr Leu Arg Ala Ser Ser Ser Ala Gly Thr Asp Pro Gin 1575 1580 1585 Leu Leu Leu Tyr Arg Val Val Arg Gly Pro Gin Leu Gly Arg Leu Phe 1590 1595 1600 His Ala Gin Gin Asp Ser Thr Gly Glu Ala Leu Val Asn Phe Thr Gin 1605 1610 1615 Ala Glu Val Tyr Ala Gly Asn He Leu Tyr Glu His Glu Met Pro Pro 1620 1625 1630 1635 Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gin Leu Ser Ser 1640 1645 1650 Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe 1655 1660 1665 Glu Ala Ala Cys Pro Gin Arg Pro Ser His Leu Trp Lys Asn Lys Gly 1670 1675 1680 Leu Trp Val Pro Glu Gly Gin Arg Ala Arg He Thr Val Ala Ala Leu 1685 1690 1695 Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gin Arg Ser Glu 1700 1705 1710 1715 His Asp Val Leu Phe Gin Val Thr Gin Phe Pro Ser Arg Gly Gin Leu 1720 1725 1730 Leu Val Ser Glu Glu Pro Leu His Ala Gly Gin Pro His Phe Leu Gin 1735 1740 1745 Ser Gin Leu Ala Ala Gly Gin Leu Val Tyr Ala His Gly Gly Gly Gly 1750 1755 1760 Thr Gin Gin Asp Gly Phe His Phe Arg Ala His Leu Gin Gly Pro Ala 1765 1770 1775 Gly Ala Ser Val Ala Gly Pro Gin Thr Ser Glu Ala ' Phe Ala He Thr 1780 1785 1790 1795 Val Arg Asp Val Asn Glu Arg Pro Pro Gin Pro Gin Ala Ser Val Pro 1800 1805 1810 Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro He Ser Arg Ala Gin Leu 1815 1820 1825 Ser Val Val Asp Pro Asp Ser Ala Pro Gly Glu He Glu Tyr Glu Val 1830 1835 1840 Gin Arg Ala Pro His Asn Gly Phe Leu Ser Leu Val Gly Gly Gly Leu 1845 1850 1855 Gly Pro Val Thr Arg Phe Thr Gin Ala Asp Val Asp Ser Gly Arg Leu 1860 1865 1870 1875 Ala Phe Val Ala Asn Gly Ser Ser Val Ala Gly He Phe Gin Leu Ser 1880 1885 1890 - 59 - Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp 1895 1900 1905 He Leu Pro Ser Ala He Glu Val Gin Leu Arg Ala Pro Leu Glu Val 1910 1915 1920 Pro Gin Ala Leu Gly Arg Ser Ser Leu Ser Gin Gin Gin Leu Arg Val 1925 1930 1935 Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu He Gin Gly 1940 1945 1950 1955 Pro Gin Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser Ala Phe 1960 1965 1970 Ser Gin Phe Gin He Asp Gin Gly Glu Val Val Phe Ala Phe Thr Asn 1975 1980 1985 Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala Leu Ala Arg Gly 1990 1995 2000 Val Asn Ala Ser Ala Val Val Asn Val Thr Val Arg Ala Leu Leu His 2005 2010 2015 Val Trp Ala Gly Gly Pro Trp Pro Gin Gly Ala Thr Leu Arg Leu Asp 2020 2025 2030 2035 Pro Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr Gly Ser Val 2040 2045 2050 Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His Gly Arg Val Val Arg 2055 2060 2065 Val Pro Arg Ala Arg Thr Glu Pro Gly Gly Ser Gin Leu Val Glu Gin 2070 2075 2080 Phe Thr Gin Gin Asp Leu Glu Asp Gly Arg Leu Gly Leu Glu Val Gly 2085 2090 2095 Arg Pro Glu Gly Arg Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu 2100 2105 2110 2115 Glu Leu Trp Ala Gin Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe 2120 2125 2130 Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu 2135 2140 2145 Ser Val Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser 2150 2155 2160 Thr Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala 2165 2170 2175 Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe Ser 2180 2185 2190 2195 Val He He Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu He Leu 2200 2205 2210 Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly Lys His Asp 2215 2220 2225 Val Gin Val Leu Thr Ala Lys Pro Arg Asn Gly Leu Ala Gly Asp Thr 2230 2235 2240 Glu Thr Phe Arg Lys Val Glu Pro Gly Gin Ala He Pro Leu Thr Ala 2245 2250 2255 Val Pro Gly Gin Gly Pro Pro Pro Gly Gly Gin Pro Asp Pro Glu Leu 2260 2265 2270 2275 Leu Gin Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly Gin Tyr 2280 2285 2290 Trp Val (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..27 (D) OTHER INFORMATION: /produc = "Primer P24" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 CCTCCAGGTG GTTCTCACCG AAGAAGG (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /produc = "Primer RA 14" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 CCAGACGCCC AACCCGCCAC GATG (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /product= "Primer P23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 CTCTGTGTGG TGAGTGTAAA CTCC (2) INFORMATION FOR SEQ ID NO: 6: -63 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..27 (D) OTHER INFORMATION: /produc = "Primer RA2 " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 CCGCCACGAT GCTTCTCAGC CCGGGAC (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /product= "Primer RA12" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGCCTTGTTG GTCAGATCTA CAGC (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer Pi9" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8; CGGTTCACCA CGTCCAGGAG G (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ; ix ) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /product= "Primer RA9" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 CTCACTGGCT GCCTTCCCTG CCTG (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer RM11" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 GTCAGTGCTC AGGTACCTCA C (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_fea ure (B) LOCATION:1..24 (D) OTHER INFORMATION:/product= "Primer RA5" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 CACGGCGAGC TGGAGCTAGA CATC (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer P9' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 GCCACGTCGT CTGTAGTCAG C (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..21 (D) OTHER INFORMATION: /product= "Primer P10" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 GACTCCGCTG ATGGTCTGCA C (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:!..24 (D) OTHER INFORMATION: /product= "Primer P7" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GCAGGTCCTG CTGGGAGAAG GCTG (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer P6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GTCGAGGTTG GTGTCCAGGT G (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATIO :1..23 (D) OTHER INFORMATION:/product= "Primer P3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 GCTCTTCCAC CATTCTCCAG GAG (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..22 (D) OTHER INFORMATION: /product= "Primer P4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 GAGGGTCCTG GCTTGAGGTC CG (2) INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..20 (D) OTHER INFORMATION: /product= "Primer BA50" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 GTCCTGCTGG GCGTGGAACA (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..18 (D) OTHER INFORMATION: /product= "Primer BA51" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GAACAGCCGG CCTAGCTG (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..33 (D) OTHER INFORMATION: /product= "Primer RMP16" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 AGTGAATTCG ATGCAGTCCG GCCGCGGCCC CCC (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ( ix ) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /product= "Primer RMP19" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 CCAGAGAGTG GGGCCCAGAG AAGC (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..25 (D) OTHER INFORMATION: /product= "Primer RMP17" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GGAGAAAGCT TCATAATGGC CATAG (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..23 (D) OTHER INFORMATION:/product= "Primer RMP20" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 GGGCTTCTCT GGGCCCCACT CTC (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer RMPll' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 GTCAGTGCTC AGGTACCTCA C (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..25 (D) OTHER INFORMATION: /product= "Primer RMP18" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25 CTATGGCCAT TATGAAGCTT TCTCC (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:!..20 (D) OTHER INFORMATION: /produc = "Primer RMP9" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GCAGCTGGAT CCAGCCACGG (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid Yx) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /product= "Primer RMP10" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27 GTGAGGTACC TGAGCACTGA C (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..20 (D) OTHER INFORMATION: /product= "Primer RMP8" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28 CCGTGGCTGG ATCCAGCTGC (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..21 (D) OTHER INFORMATION: /produc = "Primer RMP12" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29 GGCTCCAGCA CCTGCAGGCT G (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid Yx) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..22 (D) OTHER INFORMATION: /product= "Primer RMP14" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30 GAGGCAGCTG AGATCAGAAG GG (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..24 (D) OTHER INFORMATION: /product= "Primer RMP15" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GACCTTTGTT CTTCCAGAGG TGGC (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:!..22 (D) OTHER INFORMATION: /product= "Primer RMP3 " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32 CCAAAGCTTG GGGCACCTCC AG (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid ( ix ) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION: 1..20 (D) OTHER INFORMATION: /product= "Primer RMP4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 CCCTAGAGCT CCAGCTGTCC (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATIO :1..30 (D) OTHER INFORMATION: /product= "Primer RMP1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 CTGGAATTCT TAAGCCTGCC TCCACCTTGG (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATIO :1..22 (D) OTHER INFORMATION: /product= "Primer RMP2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35 CTGGAGGTGC CCCAAGCTTT GG

Claims: Claims 1. Isolated protein designated melanoma-associated chondroitin sulfate proteoglycan (MCSP) and having substantially the amino acid sequence of the mature protein as set forth in SEQ ID NO:2 or an amino acid mutant of said protein excluding the deletional amino acid mutant with the amino acid sequence extending from the amino acid at position 1594 to the amino acid at position 2293 in SEQ ID NO:2, or a derivative of said protein or mutant, particularly such protein, amino acid mutant, or derivative thereof, which is in a suitably immunogenic form. 2. A method for preparing the protein, mutant or derivative according to claim 1 comprising isolation from a natural source, chemical synthesis and/or recombinant DNA technology. 3. A method for the generation of an antibody which specifically binds to MCSP, said method comprising the step of administering to a mammal a protein or mutant, or a derivative thereof, according to claim 1. 4. A pharmaceutical composition comprising a protein or mutant, or a derivative thereof, according to claim 1, and a pharmaceutically acceptable carrier. 5. Nucleic acid comprising an isolated nucleic acid coding for a protein according to claim 1, preferably such nucleic acid which is a DNA. 6. A method for identifying a nucleic acid encoding MCSP, or a novel non-human homologue thereof, comprising contacting a sample comprising candidate DNA or RNA with a nucleic acid comprising at least 14 contiguous bases that are the same as (or complementary to) any 14 or more contiguous bases set forth in SEQ ID NO: 1, and identifying nucleic acid(s) which hybridize (s) to said probe. 7. A host cell capable of producing the protein, amino acid mutant, or derivative thereof according to claim 1, and containing a heterologous nucleic acid coding for said protein or mutant, or derivative thereof. 8. The protein, mutant or derivative according to claim 1 for use in the prophylactical or therapeutical treatment of the human body, particularly for use in the control or (adjuvant) treatment of a MCSP-expressing tumor, such as melanoma, sarcoma and glioblastoma. 9. Use of the protein, mutant or derivative according to claim 1 , or a composition comprising said protein, mutant or derivative for the manufacture of a medicament, particularly a medicament suitable for the control or (adjuvant) treatment of a MCSP-expressing tumor, such as melanoma, sarcoma and glioblastoma. 10. A method for identifying a compound which is capable of interacting with the protein, amino acid mutant, or derivative thereof according to claim 1, comprising contacting said protein, mutant or derivative, or a composition of matter comprising said protein, mutant, or derivative, with at least one compound to be tested for its ability to interact with said protein, mutant, or derivative, wherein a change of the biological activity of the protein, mutant, or derivative is indicative of the interaction. 11. Nucleic acid according to claim 5, wherein the isolated nucleic acid is a DNA having substantially the nucleotide sequence set forth in SEQ ID NO: 1 , or a fragment thereof excluding the fragment with the sequence extending from bp 4867 to bp 7898 in SEQ ID NO:l and the DNA with the sequence extending from bp 4858 to 5357 in SEQ ID NO:l, respectively.. 12. Nucleic acid according to claim 5, which is a hybrid vector. 13. The amino acid mutant according to claim 1, which is a deletional amino acid mutant, or a derivative thereof. 14. A method of vaccinating a human in need thereof comprising the step of administering to said human a protein or mutant, or a derivative thereof, according to claim 1. 15. A test kit for the qualitative or quantitative determination of anti-MCSP antibodies comprising a protein or mutant, or a derivative thereof, according to claim 1.

Inventors: PLUSCHKE GERD, SCHMID PETER, PLUSCHKE, GERD, SCHMID, PETER
Applicants: CIBA GEIGY AG, PLUSCHKE GERD, SCHMID PETER, NOVARTIS AG, PLUSCHKE, GERD, SCHMID, PETER
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61K39, C07K14, C12N15