MELANOMA ANTIGENS P15 AND THEIR APPLICATION IN DIAGNOSTIC AND THERAPEUTIC PROCEDURES
This invention is in the field of prevention and treatment of human cancers. More specifically, this invention relates to the p15 gene which encodes melanoma antigens recognized by T-Cells and their corresponding proteins and to preventative, diagnostic and therapeutic applications which employ these genes, proteins or peptides. Melanomas are aggressive, frequently metastatic tumors derived from either melanocytes or melanocyte related nevus cells (" T cells play an important role in tumor regression in most murine tumor models. Tumor infiltrating lymphocytes (TIL) that recognize unique cancer antigens can be isolated from many murine tumors. The adoptive transfer of these TIL plus interleukin-2 can mediate the regression of established lung and liver metastases ( T cell receptors on CD8+ T cells recognize a complex consisting of an antigenic peptide (9-10 amino acids for HLA-A2), β-2 microglobulin and class I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C, in humans). Peptides generated by digestion of endogenously synthesized proteins are transported into the endoplastic reticulum, bound to class I MHC heavy chain and β2 microglobulin, and finally expressed in the cell surface in the groove of the class I MHC molecule. Therefore, T cells can detect molecules that originate from proteins inside cells, in contrast to antibodies that detect intact molecules expressed on the cell surface. Therefore, antigens recognized by T cells may be more useful than antigens recognized by antibodies. Strong evidence that an immune response to cancer exists in humans is provided by the existence of lymphocytes within melanoma deposits. These lymphocytes, when isolated, are capable of recognizing specific tumor antigens on autologous and allogeneic melanomas in an MHC restricted fashion. ( Peripheral blood lymphocytes have been used to identify potential melanoma tumor antigens. This invention relates, in general, to a nucleic acid sequence, encoding melanoma antigens recognized by T-lymphocytes and protein and peptides encoded by these sequences. This invention further provides bioassays for these nucleic acid sequences, proteins and peptides. This invention also provides therapeutic uses for the nucleic acid sequences, proteins or peptides described herein. It is a general object of the present invention to provide a substantially purified and isolated nucleic acid sequence which encodes for the p15 melanoma antigen. In particular, the present invention relates to the following: For the purpose of a more complete understanding of the invention, the following definitions are described herein. Nucleic acid sequences includes, but is not limited to, DNA, RNA or cDNA. Nucleic acid sequence as used herein refers to an isolated nucleic acid sequence. p15 messenger RNA (mRNA) refers to one or more RNA transcripts which are a product of the p15 gene. Substantially homologous as used herein refers to substantial correspondence between the nucleic acid sequence of p15 shown in Figure 1 (SEQ ID NO: 1) and that of any other nucleic acid sequence. Substantially homologous means about 50-100% homologous homology, preferably by about 70-100% homology, and most preferably about 90-100% homology between the p15 sequence and that of any other nucleic acid sequence. In addition, substantially homologous as used herein also refers to substantial correspondences between the amino acid sequence of the p15 antigen shown in Figure 1 (SEQ ID NO: 2) and that of any other amino acid sequence. Major Histocompatibility Complex (MHC) is a generic designation meant to encompass the histo-compatibility antigen systems described in different species including the human leucocyte antigens (HLA). The term melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocytes related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma insitu, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. Such melanomas in mammals may be caused by, chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, and presentation on a cell, or carcinogenic agents. The aforementioned melanomas can be diagnosed, assessed or treated by methods described in the present application. By atypical mole we mean a mole with features that are abnormal and may be precancerous. By melanoma antigen or immunogen we mean all or parts thereof of the p15 protein or peptides based on the p15 protein sequence capable of causing a cellular or humoral immune response in a mammal. Such antigens may also be reactive with antibodies from animals immunized with all, part or parts of the p15 protein (Figure 1; SEQ ID NO: 2). Such a protein or peptide may be encoded by all or part of the p15 nucleic acid sequence of this invention. By immunogenic peptide we mean a peptide derived from the p15 protein sequence (FIGURE 1; SEQ ID NO: 2) or the tyrosinase peptides (SEQ ID NO: 7 and SEQ ID NO: 8) capable of causing a cellular or humoral immune response in a mammal. Such peptides may also be reactive with antibodies from an animal immunized with the peptides. Such peptides may be about 5-20 amino acid in length preferably about 8 to 15 amino acids in length, and most preferably about 9-10 amino acids in length. One skilled in the art will understand that the bioassays of the present invention may be used in the analysis of biological samples or tissues from any vertebrate species. In a preferred embodiment, mammalian biological samples or tissues are analyzed. Tissue includes, but is not limited to, single cells, whole organs and portions thereof. Biological samples include, but are not limited to, tissues, primary cultures of mammalian tissues, biopsy specimens, pathology specimens, and necropsy specimens. Mammal includes but is not limited to, humans, monkeys, dogs, cats, mice, rats, pigs, cows, pigs, horses, sheep and goats. The present invention provides a nucleic acid sequence which encodes a novel melanoma antigen recognized by T cells. The gene encoding this novel melanoma antigen is designated p15. The p15 cDNA shows no significant homology to any known melanoma antigen or protein and thus represents a gene encoding a new melanoma antigen. The only long open reading frame in this cDNA encodes a 128 amino acid polypeptide with a molecular weight (MW) of approximately 15 kilodaltons (kd) beginning with the first in frame methionine. p15 does not appear to contain any features which would identify it as a member of any known gene family, and lacks a conventional leader sequence, as well as consensus sites for N-linked glycosylation and any extended hydrophobic domains. p15 RNA is expressed in cultured melanoma and melanocyte cell lines and a wide variety of human tissues such as retina, testis, and brain. The cDNA sequence for p15 is shown in Figure 1 (SEQ ID NO: 1), the deduced amino acid sequence for the p15 protein is also shown in Figure 1 (SEQ ID NO: 1). The nucleic acid sequence for p15 shown in Figure 1 (SEQ ID NO: 1), represents a preferred embodiment of the invention. It is, however, understood by one skilled in the art that due to the degeneracy of the genetic code variations in the cDNA sequence shown in Figure 1 (SEQ ID NO: 1) will still result in a DNA sequence capable of encoding the p15 protein antigen. Such DNA sequences are therefore functionally equivalent to the sequence set forth in Figure 1 (SEQ ID NO: 1). Further, a person of skill in the art will understand that there are naturally occurring allelic variations in a given species of the p15 nucleic acid sequence shown in Figure 1 (SEQ ID NO: 1). Also described herein are nucleic acid sequences which are complimentary to nucleic acid sequences capable of hybridizing to the p15 nucleic acid sequence shown in Figure 1 under low stringency conditions. One of skill in the art will understand what it is meant by low stringency conditions and the modifications necessary to obtain low stringency conditions. Elements that can be varied to effect stringency include, but are not limited to, salt concentrations or temperature.( This invention further includes p15 protein or peptides or a substitution mutant shown in SEQ ID NO:23 having substantially the same function as the p15 antigen or protein of this invention. Such proteins or polypeptides include, but are not limited to, a fragment of the protein, or a substitution, addition or deletion mutant of the p15 protein. Also described herein are proteins or peptides that are substantially homologous to the p15 antigen. The term "analog" includes any polypeptide having an amino acid residue sequence substantially identical to the p15 sequence specifically shown herein (Figure 1; SEQ ID NO: 2) in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the p15 antigen as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid or another. The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue. "Chemical derivative" refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Examples of such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Proteins or polypeptides described herein also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is encoded is the DNA of p15, so long as the requisite activity is maintained. This invention also provides a recombinant DNA molecule comprising all or part as defined in the appended claims of the p15 nucleic acid sequence (SEQ ID NO: 1) and a vector. Expression vectors suitable for use in the present invention comprise at least one expression control element operationally linked to the nucleic acid sequence. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional preferred or required operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system. It will be understood by one skilled in the art the correct combination of required or preferred expression control elements will depend on the host system chosen. It will further be understood that the expression vector should contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods ( Another aspect of this invention relates to a non-human host organism into which recombinant expression vector containing all or part as defined in the appended claims of the p15 nucleic acid sequence has been inserted. The host cells transformed with the p15 nucleic acid sequence of this invention includes eukaryotes, such as animal, plant, insect and yeast cells and prokaryotes, such as E. coli. The means by which the vector carrying the gene may be introduced into the cell include, but are not limited to, microinjection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art ( In a preferred embodiment, eukaryotic expression vectors that function in eukaryotic cells are used. Examples of such vectors include, but are not limited to, retroviral vectors, vaccinia virus vectors, adenovirus vectors, herpes virus vector, fowl pox virus vector, plasmids, such as pCDNA3 (Invitrogen, San Diego, CA) or the baculovirus transfer vectors. Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, NIH/3T3 cells, 293 cells (ATCC# CRL1573), T2 cells, dendritic cells, or monocytes. In a preferred embodiment the recombinant p15 protein expression vector is introduced into mammalian cells, such as NIH/3T3, COS-7, CHO, 293 cells (ATCC #CRL 1573), T2 cells, dendritic cells, or monocytes to ensure proper processing and modification of the p15 protein. By way of example, the p15 cDNA is introduced into COS7 cells ( In one embodiment the expressed recombinant p15 protein may be detected by methods known in the art which include Coomassie blue staining and Western blotting using antibodies specific for the p15 protein. In a further embodiment, the recombinant protein expressed by the host cells can be obtained as a crude lysate or can be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ionexchange chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography and the like. ( The nucleic acid sequence or portions thereof, of this invention are useful as probes for the detection of expression of the p15 gene in normal and diseased tissue. Therefore, another aspect of the present invention relates to a bioassay for detecting messenger RNA encoding the p15 protein in a biological sample comprising the steps of (a) contacting a biological sample with all or part of the nucleic acid sequence of this invention under conditions allowing a complex to form between said nucleic acid sequence and said messenger RNA, and (b) detecting said complexes. This method may further comprise a step (c) of determining the level of said messenger RNA. RNA can be isolated as whole cell RNA or as poly(A)+ RNA. Whole cell RNA can be isolated by a variety of methods known to those skilled in the art. ( Detection of complexes in Step (b) of the bioassay can also be carried out by a variety of techniques. Detection of the complexes by signal amplification can be achieved by several conventional labelling techniques including radiolabels and enzymes ( Examples of biological samples that can be used in this bioassay include, but are not limited to, primary mammalian cultures, continuous mammalian cell lines, such as melanocyte cell lines, mammalian organs such as skin or retina, tissues, biopsy specimens, neoplasms, pathology specimens, and necropsy specimens. In a preferred embodiment, a32P radiolabelled p15 probe, as exemplified in Example 1, is used. The approximately 0.9 Kilobase (kb) cDNA (Figure 1; SEQ ID NO: 1) was cloned into the pCDNA3 vector and the resulting plasmid, deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, MD 20852, USA on January 9, 1995, ATCC Deposit No. 97015. The full length p15 nucleic acid sequence can be isolated from the pCDNA3 plasmid by digestion with BstXI and NotI restriction enzymes. This 0.9kb nucleic acid sequence can then be used as a probe. This probe is used to detect p15 mRNA in total RNA or poly A+ RNA isolated from a variety of tissues or biological samples. Alternatively the p15 probe is the 462 base pair BamHI/Pst I fragment from the p15 gene (Figure 1; SEQ ID NO: 1; nucleic acids 15 to 476). In another embodiment, combinations of oligonucleotide pairs based on the p15 sequence in Figure 1 (SEQ ID NO: 1) are used as Polymerase Chain Reaction (PCR) primers to detect p15 mRNA in a biological sample. These primers can be used in a method following the reverse transcriptase - Polymerase Chain Reaction (RT-PCR) process for amplifying selected RNA nucleic acid sequences as detailed in The p15 nucleic acid sequence or portions thereof (Figure 1: SEQ ID NO: 1) of this invention are useful to detect p15 genomic DNA or alterations of the p15 gene in normal or diseased mammalian tissue. By alteration, we mean additions, deletions, substitutions, rearrangements or duplications in the p15 gene sequence or gene amplification of the p15 gene sequence. Therefore, another aspect of the present invention relates to an assay for detecting the p15 genomic DNA or alterations of the p15 gene in a biological sample. Such an assay may comprise the steps of (a) contacting all or part of the nucleic acid sequence of this invention with genomic DNA isolated from a biological sample under conditions allowing a complex to form between said nucleic acid sequence and said genomic DNA, and (b) detecting said complexes. Determining alterations in said p15 gene can be performed by comparison to a control sample or other conventional methods. Standard methods for isolating DNA from a biological sample, detecting alterations in a gene and detecting complex between the p15 nucleic acid probe and genomic DNA sequences are provided in manuals such as All or parts of the p15 nucleic acid sequences of this invention (Figure 1; SEQ ID NO: 1) can also be used as probes to isolate the p15 homologs in other species. In a preferred embodiment the p15 cDNA (Figure 1; SEQ ID NO: 1) is used to screen a mammalian cDNA library; positive clones are selected and sequenced. Examples of tissue sources from which the cDNA library can be synthesized include, but are not limited to skin, retina, melanocytes, neonatal brain, testes and skin. Preferably a melanoma library is screened using the p15 nucleic acid sequences as a probe (Figure 1; SEQ ID N0: 1). One skilled in the art will understand the appropriate hybridization conditions to be used to detect the homologs. Conventional methods for nucleic acid hybridization, construction of libraries and cloning techniques are described in We have determined that all or parts thereof of the p15 protein is an antigen present on melanoma cells. It is therefore another aspect of this invention to provide p15 nucleic acid probes to be utilized in detecting p15 RNA or alterations in the level of p15 mRNA in biological sample isolated from a mammal afflicted with a disease. Examples of such diseases include, but are not limited to, melanomas. By alterations in the level of p15 mRNA we mean an increase or decrease in the level of an RNA relative to a control sample or the appearance or disappearance of the p15 mRNA relative to a control sample. Detection in the alterations of p15 mRNA may allow for diagnosis or the assessment of the diseased state. Therefore, alterations in the level of p15 mRNA may be predictive of the prognosis for the afflicted mammal. In another embodiment all or parts thereof of the nucleic acid of this invention can be used in in situ hybridization on mammalian tissues to determine the precise site or subcellular site of expression of the p15 gene within a tissue. A preferred method of labeling the p15 nucleic acid sequence is synthesizing a35S - labeled RNA probe by in vitro transcription utilizing polymerases known to those skilled in the art. Conventional methods for preparation of tissues for In yet another embodiment of this invention the p15 (SEQ ID NO: 1 nucleic acid sequence can be used to generate non-human transgenic animals. Preferably the p15 gene is introduced into an animal or an ancestor of the animal at an embryonic stage, preferably at the one cell stage and generally not later than about the eight cell stage. There are several means by which non-human transgenic animals carrying a p15 gene can be made. One method involves the use of retroviruses carrying all or part of the p15 sequence. The retroviruses containing the transgene are introduced into the embryonic animal by transfection. Another methods involves directly injecting the transgene into the embryo. Yet another methods employs the embryonic stem cell method or homologous recombination method known to workers in the field. Examples of animals into which the p15 transgene can be introduced include, but are not limited to, non-human primates, mice, rats or other rodents. Such transgenic animals may be useful as biological models for the study of melanoma and to evaluate diagnostic or therapeutic methods for melanoma. This invention further comprises an antibody or antibodies reactive with the p15 protein or peptides having the amino acid sequence defined in Figure 1 (SEQ ID NO: 2) or a substitution mutant as defined in the claims. In this embodiment of the invention the antibodies are monoclonal or polyclonal in origin. p15 protein or peptides used to generate the antibodies may be from natural or recombinant sources or generated by chemical synthesis. Natural p15 proteins can be isolated from mammalian biological samples. Biological samples include, but is not limited to mammalian tissues such as fresh melanoma, skin, retina, primary or continuous cultures of mammalian cells such as melanoma cultures or cultured melanocytes and normal tissues such as fibroblasts. The natural p15 proteins may be isolated by the same methods described above for recombinant proteins. Recombinant p15 proteins or peptides may be produced and purified by conventional methods. Synthetic p15 peptides may be custom ordered or commercially made based on the predicted amino acid sequence of the present invention (Figure 1; SEQ ID NO: 2) or synthesized by methods known to one skilled in the art ( Exemplary antibody molecules for use in the detection methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules or those portions of an immunoglobulin molecule that contain the antigen binding site, including those portions of immunoglobulin molecules known in the art as F(ab), F(ab'); F(ab')2 and F(v). Polyclonal or monoclonal antibodies may be produced by methods known in the art. ( The antibodies of this invention may react with native or denatured p15 protein or peptides or analogs thereof. The specific immunoassay in which the antibodies are to be used will dictate which antibodies are desirable. Antibodies may be raised against the p15 protein or portions thereof or against synthetic peptides homologous to the p15 amino acid sequence. In one embodiment the antibodies of this invention are used in immunoassays to detect the novel p15 protein in biological samples. In this method the antibodies of the present invention are contacted with a biological sample and the formation of a complex between the p15 antigen and antibody is detected. Immunoassays of the present invention may be radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. (In " The antibodies of this invention can be used in immunoassays to detect p15 antigen or alteration in the level of expression of the p15 antigen in biological samples isolated from mammals afflicted with a disease or disorder. Examples of biological samples include, but are not limited to, mammalian tissues, biopsy tissue samples, melanoma and lymph node biopsy samples, pathology and tissue samples. Examples of diseases that can be assessed by these immunoassays, include, but are not limited to, melanomas and tissues which are secondary sites for melanoma metastasis. By alteration in level of expression, we mean an increase or decrease of the p15 protein or portions thereof relative to a control sample. Alteration is also meant to encompass substitution, deletion, rearrangement or addition mutants of the p15 protein as well as the presence of the p15 protein or portions thereof in the wrong cellular compartment. Such mutations can be determined by using the antibodies of this invention known to react with specific epitopes of the p15 protein and determining which epitopes are present relative to a control. The antibodies of this invention can therefore be used in an immunoassay to diagnose, assess or prognoses a mammal afflicted with the disease. In a preferred embodiment, the p15 antibodies of this invention are used to assess the presence of the p15 antigen from a tissue biopsy of a mammal afflicted with melanoma using immunocytochemistry. Such assessment of the delineation of the p15 antigen in a diseased tissue can be used to prognose the progression of the disease in a mammal afflicted with the disease. Specifically the p15 antibodies can be used to characterize the radial and vertical growth phases of the melanoma lesion. Conventional methods for immunohistochemistry are described in ( In another embodiment, antibodies of this invention may be used to purify the p15 protein or portions thereof. Immunoaffinity chromatography can be performed by conventional methods known to one skilled in the art ( In another embodiment rabbit antisera containing antibodies which specifically recognize the p15 protein is used to detect said protein in Western Blot Analysis. Such antisera is directed to all, or a part or parts of the p15 protein or synthetic peptides derived from the p15 protein sequence. Preferably a p15 synthetic peptide derived from the p15 predicted amino acid sequence is used (Figure 1; SEQ ID NO: 2). The peptide is synthesized by standard methods on an automated peptide synthesizer and purified by high pressure liquid chromatography (HPLC) as described in Example 1. The purified peptide may be conjugated to a carrier as described in ( The recombinant or natural p15 protein, peptides, or analogs thereof may be used as a vaccine either prophylactically or therapeutically. When provided prophylactically the vaccine is provided in advance of any evidence of melanoma. The prophylactic administration of the p15 vaccine should serve to prevent or attenuate melanoma in a mammal. Mammals, preferably human, at high risk for melanoma can be prophylactically treated with the vaccines of this invention. Examples of such mammals include, but are not limited to, humans with a family history of melanoma, humans with a history of atypical moles, humans with a history of FAM-M syndrome or humans afflicted with melanoma previously resected and therefore at risk for reoccurrence. When provided therapeutically, the vaccine is provided to enhance the patient's own immune response to the tumor antigen present on the melanoma or metastatic melanoma. The vaccine, which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector, cell lysates from cells transfected with a p15 recombinant expression vector, or a culture supernatant containing the expressed protein. Alternatively, the immunogen is a partially or substantially purified recombinant p15 protein, peptide or analog thereof. The proteins or peptides may be conjugated with lipoprotein or administered in liposomal form or with adjuvant using conventional methodologies. While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation. Formulations suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials. The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, anti-adsorption agent may be used. Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the p15 protein, peptides and analogs thereof into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others. The proteins of the present invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above. Vaccination can be conducted by conventional methods. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier to make the protein immunogenic. Examples of such carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like. The immunogen also may be coupled with lipoproteins or administered in liposomal form or with adjuvants. The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like. The immunogen may be administered once or at periodic intervals until a significant titer of anti-p15 immune cells or anti-p15 antibody is produced. The presence of anti-p15 immune cells may be assessed by measuring the frequency of precursor CTL (cytoxic T-lymphocytes) against p15 antigen prior to and after immunization by a CTL precursor analysis assay ( The administration of the vaccine or immunogen of the present invention may be for either a prophylactic or therapeutic purpose. When provided prophylactically, the immunogen is provided in advance of any evidence or in advance of any symptom due to melanoma. The prophylactic administration of the immunogen serves to prevent or attenuate melanoma in a mammal. When provided therapeutically, the immunogen is provided at (or shortly after) the onset of the disease or at the onset of any symptom of the disease. The therapeutic administration of the immunogen serves to attenuate the disease. By way of example, a vaccine prepared using recombinant p15 protein or peptide expression vectors may be used. To provide a vaccine to an individual a genetic sequence which encodes for all or part of the p15 nucleic acid sequence is inserted into a expression vector, as described above, and introduced into the mammal to be immunized. Examples of vectors that may be used in the aforementioned vaccines include, but are not limited to, defective retroviral vectors, adenoviral vectors, vaccinia viral vectors, fowl pox viral vectors, or other viral vectors ( Alternatively all or parts thereof of a substantially or partially purified the p15 protein may be administered as a vaccine in a pharmaceutically acceptable carrier. By way of example, ranges of p15 protein to be administered may be 0.001 to 100 mg per patient, preferred doses are 0.01 to 100mg per patient. In a preferred embodiment, the p15 peptides AYGLDFYIL (p1510-18; SEQ ID NO: 5) or EAYGLDFYIL (p159-18; SEQ ID NO: 6) (presented in single letter code) or analogs thereof are administered therapeutically or prophylactically to a mammal in need of such treatment. By way of example, doses may be 0.001 mg to 100 mg, preferred doses are 0.01 mg to 100 mg. The peptide may be synthetically or recombinantly produced. Immunization is repeated as necessary, until a sufficient titer of anti-immunogen antibody or immune cells has been obtained. In yet another alternative embodiment a viral vector, such as a retroviral vector, can be introduced into mammalian cells. Examples of mammalian cells into which the retroviral vector can be introduced include, but are not limited to, primary mammalian cultures or continuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC #CRL 1573). The means by which the vector carrying the gene may be introduced into a cell includes, but is not limited to, microinjection, electroporation, transfection or transfection using DEAE dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art ( The vaccine formulation of the present invention comprise an immunogen that induces an immune response directed against the melanoma associated antigens such as the melanoma associated p15 antigen. The vaccine formulations may be evaluated first in animal models, or in nonhuman primates and finally in humans. The safety of the immunization procedures is determined by looking for the effect of immunization on the general health of the immunized animal (weight change, fever, appetite behavior etc.) and looking for pathological changes on autopsies. After initial testing in animals, melanoma cancer patients can be tested. Conventional methods would be used to evaluate the immune response of the patient to determine the efficiency of the vaccine. In yet another embodiment of this invention all, part, or parts of the p15 protein or p15 peptides may be exposed to dendritic cells cultured invitro. The cultured dendritic cells provide a means of producing T-cell dependent antigens comprised of dendritic cell modified antigen or dendritic cells pulsed with antigen, in which the antigen is processed and expressed on the antigen activated dendritic cell. The p15 antigen activated dendritic cells or processed dendritic cell antigens may be used as immunogens for vaccines or for the treatment of melanoma. The dendritic cells should be exposed to antigen for sufficient time to allow the antigens to be internalized and presented on the dendritic cells surface. The resulting dendritic cells or the dendritic cell process antigens can than be administered to an individual in need of therapy. Such methods are described in T-cells isolated from individuals can be exposed to the p15 protein or portions thereof invitro and then administered to a patient in need of such treatment in a therapeutically effective amount. Examples of where T-lymphocytes can be isolated, include but are not limited to, peripheral blood cells lymphocytes (PBL), lymph nodes, or tumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolated from the individual to be treated or from a donor by methods known in the art and cultured invitro ( In addition to use as a vaccine, the compositions can be used to prepare antibodies to p15 antigen, peptides or analogs thereof. The antibodies can be used directly as anti-melanoma agents. To prepare antibodies, a host animal is immunized using the p15 protein, peptides or analogs thereof as the immunogen and bound to a carrier as described above for vaccines. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the immunogen. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-cancer agents such as chemotherapy. The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas. Humanized antibodies (i.e., nonimmunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but nonimmunogenic portion (i.e., chimeric antibodies). Such chimeric antibodies may contain the reactive or antigen binding portion of an antibody from one species and the Fc portion of an antibody (nonimmunogenic) from a different species. Examples of chimeric antibodies, include but are not limited to, non-human mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et al., International Patent Application General reviews of "humanized" chimeric antibodies are provided by Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution ( The antibodies or antigen binding fragments may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E. coli is the subject the PCT patent applications; publication number The antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at a range of about 1-100mg per patient. Thus, antibodies reactive with the p15 antigen can be passively administered alone or in conjunction with other anti-cancer therapies to a mammal afflicted with melanoma. Examples of anti-cancer therapies include, but are not limited to, chemotherapy, radiation therapy, adoptive immunotherapy therapy with TIL. Alternatively, anti p15 antigen antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-p15 antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-p15 antibodies, or by affinity chromatography using anti-p15 antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic p15-antigen and may be used to prepare an p15 melanoma antigen vaccine rather than using the p15 protein, peptides analogs or portions thereof. When used as a means of inducing anti-p15 antibodies in an animal, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously, interlesionally, or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable. The p15 derived proteins or peptides of the invention are also intended for use in producing antiserum designed for pre- or post-disease prophylaxis. Here the p15 antigen, peptides or analogs thereof is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected proteins is monitored, during a several-week period following immunization, by periodic serum sampling to detect the presence of anti-p15 serum antibodies, using an immunoassay as described herein. The antiserum from immunized individuals may be administered as a prophylactic measure for individuals who are at risk of developing melanoma. The antiserum is also useful in treating an individual afflicted with melanoma for post-disease prophylaxis. For both invivo use of antibodies to p15 antigen and anti-idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-p15 antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. ( Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal p15 antigen or peptide antibodies, the antibodies must bind to p15 antigen or peptide. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-p15 antibodies. Cells producing antibodies of the desired specificity are selected. The antibodies or chimeric antibodies described herein may also be coupled to toxin molecules radioisotopes and drugs by conventional methods ( Local administration to the afflicted site may be accomplished through means known in the art, including, but not limited to, topical application, injection, and implantation of a porous device containing cells recombinantly expressing the infusion, implantation of a porous device in which the p15 antibodies or chimeric antibodies, antibodies coupled to toxins, drugs or radiolabels or portions thereof are contained. The above described antibodies and antigen binding fragments thereof may be supplied in kit form alone, or as a pharmaceutical composition for invivo use. The antibodies may be used for therapeutic uses, diagnostic use in immunoassays or as an immunoaffinity agent to purify the p15 protein or peptides as described herein. Also described herein is a tyrosinase nucleic acid sequence and amino acid sequence (Figures 7A-7D; SEQ ID NOS. 9 and 10) and antigenic or immunogenic peptides derived from the tyrosinase protein sequence. The tyrosinase nucleic acid sequence reported herein ' (Figures 7A-7D; SEQ ID NO. 9) differs from the previously reported sequence for tyrosinase ( The immunogenic peptides derived from the tyrosinase sequence (Figures 7A-7D) represent antigenic portions of the tyrosinase protein (Figure 5) recognized by HLA-A24 restricted TIL. Examples of immunogenic peptides include, but are not limited to, AFLPWHRLF (SEQ ID NO: 7) and overlapping peptide AFLPWHRLFL (SEQ ID NO: 8). Analogs of these immunogenic peptides derived from the tyrosinase amino acid sequence are also described herein. The term analog includes any peptide which displays the functional aspects of these immunogenic peptides. The term analog also includes conservative substitution or chemical derivative of the peptides as described above. These immunogenic peptides may be synthetically or recombinantly produced in the same manner or fashion as described above for p15. The immunogenic peptides (SEQ ID NO : 7 and 8) that are described herein are derived from the tyrosinase amino acid sequence and may be used as a vaccine either therapeutically or prophylactically. When provided, prophylactically the vaccine is provided in advance of any evidence of melanoma. The prophylactic administration of these peptides should serve to prevent or attenuate melanoma in a mammal. Mammals preferably humans, at high risk for melanoma prophylactically can be treated with these vaccines. Alternatively, the vaccine may be provided therapeutically to enhance the patients own immune response to the tumor antigen prescribed on the melanoma or metastatic melanoma. The vaccine, which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector carrying a nucleic acid sequences encoding tyrosinase immunogenic peptide or a culture supernatant containing the expressed protein. Expression vectors into which nucleic acid sequences encoding these immunogenic peptides may be introduced are the same as those described above for p15. Alternatively, the immunogen is a partially or substantially purified recombinant tyrosinase peptide or analog thereof. While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as pharmaceutical compositions, formulations or preparations as described above for p15. Vaccination can be conducted by conventional methods previously described above for p15. The tyrosinase immunogenic peptides and nucleic acids sequences encoding them may be used in bioassays, or to generate antibodies in the same manner or fashion as described above for p15. In yet another embodiment of this invention, multivalent vaccines against one or more melanoma antigens are provided. Such multivalent vaccines may comprise all or part of the p15 protein or peptides or tyrosinase peptides disclosed herein or combinations thereof. Alternatively, multivalent vaccines comprising p15 protein or peptides or the immunogenic tyrosinase peptides disclosed herein may be combined with other known melanoma antigens to create a multivalent melanoma vaccine. Examples of known melanoma antigens include, but are not limited to, MART-1, gp100 MAGE-1 and MAGE-2. Once the genes or nucleic acid sequences encoding melanoma antigens are identified, the next step is to determine the antigenic portion or epitope of the protein encoded by these genes. Therefore, in yet another embodiment of this invention, a method is provided for assessing the immunogenicity of peptides derived from the predicted amino acid sequences of the p15 protein (Figure 1; SEQ ID NO: 2). The method comprises the steps of: (a) preparing a plurality of peptides based on the p15 (Figure 1; SEQ ID NO: 2) amino acid sequence; (b) incubating at least one of said peptides with a mammalian cell line; (c) exposing said mammalian cells incubated with said peptide to tumor infiltrating lymphocytes (TIL); and (d) screening for recognition of TIL with said cells incubated with said peptide. It is preferred that peptides of about 25 to 5 amino acids be used, more preferably 20 to 10 amino acids and most preferably 9-10 amino acids. Examples of cells that may be used in step (b) include, but are not limited to, T2 cells, ( T cells recognize antigen complexed with MHC Class 1 molecules. The MHC locus in all mammalian species contains numerous genes and is highly polymorphic. Different MHC molecules or haplotypes types bind different antigens. In humans the HLA complex contains the HLA-A, HLA-B and HLA-C gene loci which encode class I molecules. Lymphocytes will recognize tumor antigens on the context of HLA Class 1 molecule. If the cells containing the recombinant p15 expression vector are to be screened by the TIL but are not human cells, such as COS cells, or do not express a desired haplotype an expression vector containing an MHC Class I gene may also be introduced into the cells. This, represents yet another alternative embodiment of the invention. Cells expressing p15 or tyrosinase antigens and HLA antigens can be screened with TIL to detect the presence of tumor antigens in the context of a specific MHC Class 1 restriction type. The appropriate haplotype is determined by the haplotype of the tumor from which the library is derived. Examples of MHC Class I genes that may be used include, but are not limited to, HLA-A, HLA-B and HLA-C genes. Examples of preferred MHC specificities or restriction types include, but is not limited to HLA-A1, HLA-A2, such as the HLA-A2.1 subtype, or HLA-A24 ( The following examples illustrate various aspects of the invention and in no way intended to limit the scope thereof. Melanoma-specific CTL were grown and expanded from TIL in media containing 6000 IU of IL2 (Cetus-Oncogen Division, Chirion Corp, Emeryville, CA) as described in Construction and screening of a cDNA library prepared from melanoma 888 was carried out as previously described ( Total RNA was isolated by the guanidium isothiocyanate/cesium chloride method. Total RNA from human normal tissue was purchased from Clontech (Palo Alto, CA). 20 ug of total RNA was subject to electrophoresis on a 1% agarose formaldehyde gel, and transferred to a nylon membrane. The membrane was pre-hybridized with QuickHyb (Stratagene), and hybridized with a 462 bp BamHI/PstI fragment (Figure 1; nucleotides 15-476) from the p15 cDNA was labelled with32P using the standard random primer method. Hybridization was carried out with QuickHyb according to the manufacturer's instructions, and the membrane was washed with 0.1X SSC at 55°C for 30 minutes (min) before autoradiography. DNA sequencing was carried out using a Sequenase 2.0 kit (USB, Cleveland, OH). Database searches with the nucleotide and deduced amino acid sequences were carried out using Blast and Fasta sequence alignment algorithms Peptides were made on a Gilson AMS 422 Multiple Peptide Synthesizer using standard Fmoc chemistry. Peptides were purified on an R2 reverse phase HPLC column (PerSeptive Biosystems) using an 1% to 60% acetonitrile gradient in water containing 0.05% TFA, and were >95% pure. A TIL line grown from patient 888 in 1990, TIL 888, was previously shown to recognize melanoma in an HLA-A24 restricted manner ( The specificity of TIL 1290 and TIL 888 was then examined in a cytokine release assay (Table 2). The results indicated that both 888 and 1290 mel strongly stimulate TIL 888 and TIL 1290. Two other HLA-A24 expressing melanocytes, 928 and 1300 mel, stimulated strong cytokine release from TIL 1290 and 888, whereas two melanomas which did not express HLA-A24, 397, and 624, did not stimulate significant cytokine release from these TIL. A stable transfectant of 397 mel expressing HLA-A24 stimulated significant cytokine release from both 888 and 1290 TIL, demonstrating the restriction of the cell line. The recognition pattern of TIL 888 and 1290 was not identical, however, since an HLA-A24-expressing melanocyte line, NEHM2488, stimulated the release of low but significantly levels of GM-CSF from TIL 888 (160 pg/ml), but not TIL 1290. The TIL 1290 line was then examined for recognition of tyrosinase, as well as MART-1 and gp100, two antigens which have recently shown to be recognized by HLA-A2 restricted melanoma-specific T cells ( In order to isolate the gene encoding this antigen, pools of clones prepared from an 888 melanoma cDNA library ( This cDNA clone was sequenced and found to represent a gene not previously reported. (Figure 1). The only long open reading frame in this clone encoded a 128 amino acid polypeptide with a MW of about 15 kD beginning with the first in frame methionine. This gene did not appear to contain any features which would identify it as a member of any known gene family, and lacked a conventional leader sequence, as well as consensus sites for N-linked glycosylation and any extended hydrophobic domains. The gene's product therefore appears to represent a small cytoplasmic or nuclear protein of unknown function, and was designated p15. Northern blot analysis was then carried out to determine the pattern of expression of this gene (Figure 2). These results indicated that a variety of normal tissues expressed comparable message levels to those found in melanoma cells. The normal tissues examined included spleen, testes, thymus, liver, kidney, brain, adrenal, lung, and retinal tissue, as well as EBV B cells isolated from patient 888. The lower level of expression found in EBV B cells appeared to be due to under-loading of this sample, since a subsequent blot demonstrated that relatively similar amounts of p15 message were expressed in 1290 melanoma and EBV B cells, as well as fibroblasts isolated from patient 888. To determine if the epitope recognized by TIL 1290 resulted from a mutation of the p15 gene product, RT-PCR was used to isolate gene products expressed in 888 EBV B cells. The sequence of one of the products isolated by RT-PCR from EBV B RNA, Clone 1, was identical to the p15 sequence in the region sequenced (Figure 3), and appears to represent a full length clone. Three out of the nine clones isolated by RT-PCR from EBV B cells appeared to contain truncated inserts. The sequence of one of the truncated clones, Clone 2, appears to have resulted from a recombination between residues 199 and 738 of the p15 gene (Figure 3). Clone 2 also contained one nucleotide difference from the sequence of p15 at codon 8, resulting in substitution of asparagine for aspartic acid. Expression of the epitope recognized by TIL 1290 was then tested by transfection of the full length and truncated genes (clones 1 and 2 respectively) isolated from 888 EBV B cells. Transfection of 293-A24 cells with either construct (clone 1 or 2) was found to confer the ability to stimulate levels of cytokine release comparable to that stimulated by the original p15 cDNA clone (Table 4). This data indicated that the gene encoding this antigen was also expressed in the patient's B cells. The sequence of Clone 2, which was recognized by TIL 1290, contained only 18 amino acids of the deduced coding region identified in the p15 sequence. A motif has recently been defined for HLA-A24-binding peptides by isolating peptides from this HLA restriction element, as well as by substituting amino acids at the anchor residue positions in synthetic peptides ( To isolate additional antigens recognized by TIL 1290, screening of an additional 700 pools, containing approximately 35,000 cDNA clones was performed. A second cDNA clone was isolated which strongly stimulated TIL 1290. Partial sequencing of this clone revealed that it represented a transcript of the p15 gene, lacking only 8 base pairs from the end of the 5' untranslated region of p15. P15 may represent the predominant product recognized by TIL 1290. The gene encoding the antigen recognized by TIL 1290, p15, does not possess significant similarities to known genes. TIL 1290 failed to recognize autologous EBV B cells which had not been pulsed with peptides, and normal fibroblasts but recognized a specific melanoma antigen in the context of HLA-A24. Northern blots showed that normal tissues, including EBV B cells (Figure 2) and fibroblasts, contained significant levels of RNA encoding this protein (Figure 2). The gene encoding the p15 protein could also be isolated from EBV B cells and could confer reactivity of TIL 1290 to 293-A24 cells, suggesting that this represents a non-mutated normal gene. Patient 888 was found to have malignant melanoma, and TIL and melanoma lines, designated 888, were established in 1989. The TIL 888 was infused into the autologous patient along with IL2, and a complete remission of subcutaneous, mucosal, and lung metastases was observed ( All of the tumor antigens which have so far been described in melanoma appear to represent the products of non-mutated genes expressed in normal tissues. The proteins MART-1 and gp100, gp75 and tyrosinase are expressed in normal cultured melanocytes and are expressed in retina as well as normal skin melanocytes in vivo. Gp75 and tyrosinase have been shown to be involved in melanin synthesis. TIL 1413 cell line was generated by culturing lymphocytes obtained from tumor biopsy in AIM-V medium (Life Technologies, Inc., Gaithersburg, MD) containing 5% human AB serum and 6000 international units/ml of interleukin 2 (IL-2) (Cetus-Oncogen Division, Chiron Corp., Emeryville, CA) for 30-70 days as previously described ( Melanoma cell lines (888 mel, 938 mel, 397 mel, and 586 mel) and Epstein-Barr virus transformed B cell lines (888 EBVB) were established in our laboratory and cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) ( Tyrosinase cDNA was cloned into the BstX I site of pcDNA3 ( COS-7 cells were transfected with the plasmids containing the truncated tyrosinase cDNA and HLA-A24 cDNA by Lipofectamine methods as previously described ( Briefly, 1x105 COS-7 cells were plated in a flat-bottom 96-well microplate in Dulbecco's Modified Eagle's medium (DMEM) (Biofluids, Gaithersberg, MD) without serum. 200 ng of plasmids containing the truncated genes was then mixed with 2 mg of Lipofectamine in 100 ml of DMEM for 15-45 min, added to COS cells, and incubated for 16 h. The following day, the transfection medium was removed, cells were rinsed twice with DMEM, and 1x105 TIL was added into each well in AIM-V medium containing 60 international units/ml of IL-2. After incubation for 18 hours (H), 100 ml of supernatant was collected and assayed for GM-CSF production using a granulocyte-macrophage colony stimulating factor (GM-CSF) ELISA kit (R+D Systems, Minneapolis, MN). Peptides were synthesized by a solid phase method using a multiple synthesizer (Model AMS 422, Gilson co. Inc., Worthington, OH). The peptides were purified by HPLC on an R2 reverse phase column (Perseptive Biosystems, Cambridge, MA) with an acetonitrile gradient in 0.05% TFA/water. The identity of these peptides were confirmed by mass spectrometry. Epitope was identified by reactivity of T-cells against 888 EBVB cells preincubated with each peptide using GM-CSF release assay as described above and cytotoxicity assay as described in The TIL 1413 line isolated from HLA-A24+ patient 1413 released GM-CSF when incubated with HLA-A24+ melanoma cell lines, but not HLA-A24- melanoma cell lines or the HLA-A24+ 888 EBVB line (Table 6). In addition, TIL 1413 also weakly lysed HLA-A24+ allogenic melanoma cell line (Table 7). These studies suggested that a shared melanoma antigen could be recognized by TIL 1413 in the context of HLA-A24. Tyrosinase, a shared melanoma antigen, has previously been shown to be recognized by T cells in the context of two different class I HLA alleles, HLA-A2 and HLA-A24 ( To identify the epitope recognized by TIL 1413, multiple truncated tyrosinase cDNA clones were using an exonuclease III deletion method. Exonuclease III generated removed nucleotides in the 3' to 5' direction at the Not I site and then created unidirectional nested deletions from the 3' end of tyrosinase cDNA. After ligation and isolation, these truncated tyrosinase cDNAs were then transfected into COS-7 cells along with the HLA-A24 cDNA to determine the region encoding epitope by testing TIL reactivity to the transfected COS cells using GM-CSF release assay. By determining the sequence of the truncated cDNA clones, the region coding for the epitope was delineated between 537 bp and 683 bp of the tyrosinase cDNA gene (Figure 5). Eleven peptides within this region were synthesized based on the suggested peptide binding motifs to HLA-A24 ( The peptides T9206 (AFLPWHRLF; SEQ ID NO: 7) and T10206 (AFLPWHRLFL; SEQ ID NO: 8) were further purified and titrated in order to evaluate their relative ability to sensitize 888 EBVB cells to TIL lysis. Both peptides have a similar activity in sensitizing target cells, the maximal lysis of 888 EBVB cells pulsed with these peptides was about 40% (Figure 6). Tyrosinase, a enzyme involved in melanin synthesis, was recognized by T-cells in association with two different HLA restriction elements, HLA-A2 and HLA-A24 ( It was demonstrated that tyrosinase can be recognized by HLA-A24 restricted TIL from patient 1413 and have also identified a tyrosinase epitope recognized by TIL 1413. Tyrosinase has previously been shown to be recognized by T-cells from a patient with melanoma in association with two different HLA restriction elements, HLA-A2 and HLA-A24 ( The use of COS cells transfected with a series of truncated cDNA generated by the exonuclease III gene deletion method was used to locate region containing an immunogenic epitope of tyrosinase. In order to create unidirectional deletions, the vector was cut with Xba I and filled with α-phosphorothioate deoxynucleotides which protect the plasmid from digestion with exonuclease III. The vector was also digested with Not I, which served as the starting point for digestion. Since the deletion can be controlled by varying the time of digestion, various sizes of the truncated gene can be generated by this method and the region containing the epitopes can be narrowed. Based on titration analysis, T9206 (SEQ ID NO: 7) and T10206 (SEQ ID NO: 8) peptides sensitized target cells to lysis with similar efficiency (Figure 6). The 9-mer peptide, T9206, probably represents the naturally processed peptide on tumor cell surface, since the predominant size of peptides eluted from class I MHC molecules has been reported to be nine amino acids ( All melanoma antigens identified so far, including MAGE-1, MAGE-3, gp100, MART-1, and tyrosinase, are non-mutated self-antigens ( P-15 vaccines may be efficacious in treating mammals afflicted with melanoma. For example, p15 vaccines may be administered to individuals. Mammals can be immunized with the recombinant proteins described herein in ranges of 1mg-100mg. Alternatively mammals, preferably humans may be immunized with the p15 nucleic acid sequence inserted into a viral vector such as vaccinia virus, adenovirus or fowl pox virus. By way of example, the nucleic acid sequences encoding the p15 immunogenic peptides AYGLDFYIL (p1510-18; SEQ ID NO: 5) and EAYGLDFYIL (p159-18; SEQ ID NO: 6) can be used. A range of about 106-1011 viral particles carrying the p15 nucleic acid sequences can be administered per mammal, preferably a human. The mammals will be monitored for antibodies to the immunogen or increase in cytotoxic lymphocytes (CTL) recognizing the immunogen by conventional methods or alleviation of clinical signs and symptoms of the active disease. Specific parameters to be assessed include production of immune cells that recognize the vaccine antigen or tumor regression. Such vaccines may be administered either prophylactically or therapeutically. Mammals may also be immunized with the p15 nucleic acid sequence inserted into a retroviral vector. Suggested dose ranges of the antigen in retroviruses are 106-1011 viral particles per mammal, preferably a human. Response and efficacy of the retroviral vaccines will be assessed as described above. Alternatively, the nucleic acids corresponding to the HLA-A24 immunogenic tyrosinase peptides AFLPWHRLF (SEQ ID NO: 7) and AFLPWHRLFL (SEQ ID NO: 8) may be inserted into vectors and used as vaccines. T-lymphocytes presensitized to the melanoma antigen may be effective in therapeutically treating mammals afflicted with melanoma. The T-lymphocytes are isolated from peripheral blood lymphocytes or tumor infiltrating lymphocytes and exposed in vitro to the p15 protein or peptide. T-lymphocytes are isolated from peripheral blood or melanoma tumor suspensions and cultured invitro ( The present invention provides a nucleic acid sequence encoding a melanoma antigen recognized by T lymphocytes, designated p15. This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess or prognoses a mammal afflicted with melanoma or metastata melanoma. This invention also provides immunogenic peptides derived from the p15 melanoma antigen and a second melanoma antigen designated tyrosinase. The proteins and peptides provided can serve as an immunogen or vaccine to prevent or treat melanoma.
A nucleic acid encoding a p15 protein (SEQ ID NO: 2), a peptide, or a substitution mutant thereof shown in SEQ ID NO: 23, wherein said peptide comprises at least 8 contiguous amino acids of a p15 protein (SEQ ID NO: 2) and wherein said peptide or substitution mutant is reactive to tumor infiltrating lymphocytes.
The nucleic acid of claim 1, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1.
The nucleic acid of claim 1, wherein the peptide consists of SEQ ID NO: 5 or SEQ ID NO: 6.
A nucleic acid which is complementary to the nucleic acid of any of claims 1 to 3, or a nucleic acid which specifically hybridizes with the nucleic acid of any of claims 1 to 3 under conditions of 0.1X SSC at 55 °C for 30 minutes, wherein said nucleic acid of any of claims 1 to 3 encodes for a protein, a peptide, or a substitution mutant thereof shown in SEQ ID NO: 23, which is reactive to tumor infiltrating lymphocytes.
The nucleic acid of claim 4, wherein the nucleic acid comprises a nucleotide sequence which is complementary to SEQ ID NO: 1.
A vector containing the nucleic acid of any of claims 1 to 5.
The vector of claim 6 in which the nucleic acid is operatively linked to an expression control element.
The vector of claim 7, wherein the vector is a baculovirus vector.
A non-human host organism, or a human or non-human in vitro cell transformed or transfected with the vector according to any of claims 6 to 8.
The non-human host organism, or the human or non-human in vitro cell of claim 9, wherein said host organism or cell is a eukaryote or eukaryotic cell.
The non-human host organism, or the human or non-human in vitro cell of claim 10, wherein said eukaryote or eukaryotic cell is an insect or insect cell.
A method of recombinantly producing a p15 protein or peptide that is capable of causing a cellular or humoral immune response in a mammal, said method comprising the steps of (i) culturing the host cell of any one of claims 9 to 11 under conditions to cause expression of the protein or peptide and (ii) recovering said protein.
A p15 protein, peptide, or substitution mutant thereof encoded by the nucleic acid of any of claims 1 to 5.
The protein, peptide, or substitution mutant of claim 13, wherein the protein comprises the amino acid sequence of SEQ ID NO: 2.
The protein, peptide, or substitution mutant of claim 13, which is at least 9 to 10 amino acids in length.
The protein, peptide, or substitution mutant of claim 15, wherein said peptide comprises the amino acid sequence AYGLDVYIL (SEQ ID NO: 5) or EAYGLDFYIL (SEQ ID NO: 6).
The peptide of any one of claims 13 to 16, which is a native, synthetic or recombinant peptide.
An antibody reactive with the protein, peptide, or substitution mutant of any one of claims 13 to 17.
The antibody of claim 18, which is monoclonal or polyclonal.
An antigen binding domain of the antibody of claim 18 or 19.
A method for detecting an mRNA encoding a p15 protein (SEQ ID NO: 2) in a biological sample comprising the steps of:
(a) contacting the biological sample with at least a portion of the nucleic acid shown in Figure 1 (SEQ ID NO: 1) under conditions to allow a complex to form between said nucleic acid and said mRNA; and
(b) detecting said complex.
The method of claim 21, wherein the nucleic acid comprises nucleotides 15-476 of SEQ ID NO: 1.
A method of detecting a p15 protein (SEQ ID NO: 2) in a biological sample comprising the steps of:
(a) contacting a sample with an antibody, or antigen binding portion thereof, which specifically reacts and forms a complex with said protein; and
(b) detecting the formation of said complex.
The method of any of claims 21 to 23, wherein said sample is mammalian tissue, mammalian cells, a necropsy sample, a pathology sample or a biopsy sample.
The method of claim 23 or 24, wherein said antibody or antigen binding domain thereof is an antibody according to claim 18 and 19 or the antigen binding domain of claim 20.
A method of detecting a genomic nucleic acid encoding a p15 protein (SEQ ID NO: 2) in a biological sample comprising:
(a) contacting a biological sample with at least a portion of the nucleic acid shown in Figure 1 (SEQ ID NO: 1) under conditions to allow complexes to form between said nucleic acid and said genomic nucleic acid and
(b) detecting said complexes.
The method of claim 26, wherein the nucleic acid comprises nucleotides 15-476 of SEQ ID NO: 1.
A pharmaceutical composition comprising:
(a) a protein or peptide of any one of claims 13 to 17;
(b) a nucleic acid of any of claims 1 to 5;
(c) the vector of any of claims 6 to 8;
(d) an antibody of claim 18 or 19;
(e) an antigen binding domain of claim 20, or
(f) a cell of any of claims 9 to 11,
and an acceptable excipient, diluent or carrier.
Use of:
(a) a protein or peptide of any one of claims 13 to 17;
(b) a nucleic acid of any of claims 1 to 5;
(c) the vector of any of claims 6 to 8;
(d) an antibody of claim 18 or 19;
(e) an antigen binding domain of claim 20, or
(f) a cell of any of claims 9 to 11,
in the manufacture of a medicament for preventing or treating melanoma.
A method for assessing immunogenicity of the proteins, peptides, or substitution mutuants of any one of claims 13 to 16, said method comprising the steps of:
(a) preparing a plurality of said proteins, peptides, or substitution mutants;
(b) incubating at least one of said proteins, peptides, or substitution mutants with a mammalian cell line;
(c) exposing mammalian cells of said mammalian cell line to tumor infiltrating lymphocytes (TIL); and
(d) screening for recognition of TIL with said cells.
The method of claim 30, wherein said cells in step (b) are COS cells, T2 cells or EBV transformed B cell lines.
The protein or peptide of any of claims 13 to 17, the nucleic acid of any of claims 1 to 5, the vector of any of claims 6 to 8, the antibody of claim 18 or 19, the antigen binding domain of claim 20, or the cell of any of claims 9 to 11 for use in preventing or treating melanoma.FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
SUMMARY OF THE INVENTION
and an acceptable excipient, diluent or carrier.
in the manufacture of a medicament for preventing or treating melanoma.DESCRIPTION OF THE FIGURES
DETAILED DESCRIPTION OF THE INVENTION
Example 1
Cloning Of The p15 Gene Recognized By Melanoma Specific HLA-A24 Restricted Tumor Infiltrating Lymphocytes
1290 TCb + 65 59 888 TC + 68 70 1300 TC + 47 34 928 TC + 30 28 938 TC + 59 42 1102 TC + 11 11 1123 TC + 9 5.0 1195 TC + 11 4.0 501 TC + 5 2.0 1290 fresh melc + 19 28 1406 fresh mel + 24 12 397-A24 + 20 14 397 TC - 2 2 624 TC - 3 2 888 EBV + -3 -1 501 EBV + 3 3 K562 - 2 -1 Daudi - -2 -6 a % lysis by TIL 1290 at the indicated effector (E):(T) target ratio. All targets were lysed greater than 15% by lymphokine activated killer cells at an E:T ratio of 40:1.b TC mel, tissue culture melanoma cell line.c Fresh mel, cryopreserved, noncultured melanoma cells. COS-7 pCDNA3b 10 120 COS-7 pCDNA3+HLA-A24 10 80 COS-7 pCDNA3+tyrosinase 10 130 COS-7 tyrosinase +HLA-A24 20 1500 COS-7 MART-1c+HLA-A24 20 140 COS-7 gp100c+HLA-A24 10 120 COS-7 gp75c+HLA-A24 20 130 888 mel None 1500 3200 1290 mel None 500 800 928 mel None 10 490 1300 mel None 120 1900 397 mel None <8 <8 397-A24 mel None 110 540 624 mel None <8 <8 NEHM2488d None 10 160 NEHM680 None <8 <8 None None <8 <8 a 105 of the indicated TIL were incubated with the stimulators for 18 hours and GM-CSF releaseb COS-7 cells (5X104) were transfected with 200 ng of plasmid DNA containing the indicated tumor antigen genes or vector control with 50ng of plasmid DNA containing the appropriate restriction element.c Positive controls were carried out using TIL previously shown to recognize MART-1, gp100 and gp75. COS transfected with MART-1 plus HLA-A2, HLA-A2 alone or MART-1 alone stimulated the release of 1,800, 30 and <8 pg/ml of GM-CSF, respectively, from TIL 1235. COS transfected with gp100 plus HLA-A2, HLA-A2 alone or gp100 alone stimulated the release of 1,500, 50, and 40pg/ml of GM-CSF, respectively, from TIL 1200. COS transfected with gp75 plus HLA-A31, HLA-A31 alone or gp75 alone stimulated the release of 770, 10 and 10pg/ml of GM-CSF, respectively, from TIL 586.d NEHN 2488 and NEHM 680 represent 2 normal human melanocyte lines. 293-A24 pCDNA3b <10 60 293-A24 tyrosinase <10 750 293-A24 p15 400 60 888 mel None 1,100 5,000 1290 mel None 660 2,300 624 mel None <10 <10 None None <10 30 a Assays were carried out with 2X105 TIL as described in Materials and Methods in Example 1.b 293-A24 cells (105) were transfected with 200 ng of plasmid containing the indicated genes overnight before incubation with TIL. COS-7 p15 30 60 COS-7 HLA-A24 30 100 COS-7 p15+HLA-A24 770 1,200 COS-7 Clone1b+HLA-A24 740 690 COS-7 Clone2+HLA-A24 300 650 COS-7 β-gal+HLA-A24 40 150 888 None 3,200 1,100 None None <10 30 a The indicated genes were transfected either alone or with a plasmid containing the HLA-A24 gene into 5X104 cos cells.b An RT-PCR was carried out using RNA obtained from 888 EBV B cells with primers M2a and M2b as described in Materials and Methods in Example 1. The PCR products were cloned in pCDNA3 and tested along with the full length p15 gene for their ability to stimulate cytokine release from TIL 1290 following transfection into cos cells along with HLA-A24. p1510-18 10 910 " 1 600 " 0.1 390 " 0.01 80 " 0.001 20 p159-18 10 780 " 1 570 " 0.1 390 " 0.01 70 " 0.001 <10 None 20 888 mel 2,000 TIL alone 10 a. Peptides were incubated with 105 888 EBV B cells at the indicated concentrations for 2 hours at 37°. Following this incubation, 105 TIL 1290 were added, and 18 hours later supernatants harvested and assayed for GM-CSF using a GM-CSF Elisa Kit (R&D Company, Minneapolis, Minnesota). Example 2
Identification Of A Tyrosinase Epitope Recognized By HLA-A24 Restricted Tumor Infiltrating Lymphocytes (TIL)
888 Melb none + 865 938 Mel none + 538 586 Mel none - 56 397 Mel none - 31 888 EBVB none + 28 COS-7c none - 35 COS-7 HLA-A24 + 31 COS-7 tyrosinase - 38 COS-7 tyrosinase+HLA-A24 + 292 COS-7 β-gal+HLA-A24 + 30 a TIL in the absence of melanomas secreted <40 pg/ml GM-CSF.b 5 x 105/ml melanoma cells were incubated with TIL.c COS-7 cells were transfected as described in Example 2. 888 Mel none + 12 >1,000 938 Mel none + 8 NDb 586 Mel none - -1 ND 397 Mel none - 0 ND 888 EBVBc none + 0 89 888 EBVB T10166 + -1 93 (MFNDINIYDL) (SEQ.ID NO:9) 888 EBVB T9177 + 0 93 (VWMHYYVSM) (SEQ.ID NO:10) 888 EBVB T9180 + 2 117 (HYYVSMDAL) (SEQ.ID NO:11) 888 EBVB T10180 + 1 79 (HYYVSMDALL) (SEQ.ID NO:12) 888 EBVB T9181 + -1 144 (YYVSMDALL) (SEQ.ID NO:13) 888 EBVB T9199 + 0 133 (DFAHEAPAF) (SEQ.ID NO:14) 888 EBVB T10199 + 1 101 (DFAHEAPAFL) (SEQ.ID NO:15) 888 EBVB T9206 + 26 >1,000 (AFLPWHRLF) (SEQ.ID NO:7) 888 EBVB T10206 + 20 >1,000 (AFLPWHRLFL) (SEQ.ID NO:8) 888 EBVB T10209 + 0 153 (PWHRLFLLRW) (SEQ.ID NO:16) 888 EBVB T10213 + 1 81 (LFLLRWEQEI) (SEQ.ID NO:17) a % of lysis by TIL 1413 was shown at effector (E): target (T) ratio of 40 :1.b Not determined in this experiment.c EBVB, Epstein-Barr virus-transformed cells. 888 EBVB was typed as HLA-A1 and HLA-A24. Example 4
P-15 Vaccines As A Treatment For Melanoma In Mammals
Example 5
Use Of Lymphocytes Sensitized To Immunogenic Peptides Derived From Melanoma Antigens For Therapeutically Treating Mammals Afflicted With Melanoma
SEQUENCE LISTING