WO2001074847A2 - T-cell epitope of mage-12 and related nucleic acids, vectors, cells, compositions and methods of inducing an immune response to cancer - Google Patents

Abstract

The present invention provides an isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL, an isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding the amino acid sequence VRIGHLYIL, such as SEQ ID NO: 2, a vector comprising and expressing such an isolated and purified nucleic acid molecule and a cell comprising such a vector. The present invention further provides a composition comprising the isolated and purified polypeptide or the vector in an amount effective to induce an immune response to the polypeptide in a mammal as well as a method of inducing an immune response to a cancer in a mammal having cancer, such as melanoma. The method comprises administering the composition to the mammal, which preferably has HLA-Cw*0702, in an amount effective to induce an immune response to the cancer in the mammal. The method can further comprise administering a cytokine, such as interleukin-2.

Description

T-CELL EPITOPE OF MAGE-12 AND RELATED NUCLEIC ACIDS,

VECTORS, CELLS, COMPOSITIONS AND METHODS OF

INDUCING AN IMMUNE RESPONSE TO CANCER

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL and related nucleic acids, vectors and cells as well as compositions comprising the same and methods of using the same to induce an immune response to cancer. .

BACKGROUND OF THE INVENTION The MAGE family of genes encodes human tumor rejection antigen precursors. Various genes of the MAGE family are expressed in tumor cells. Studies of the MAGE family of genes have revealed that a particular nonapeptide of MAGE- 1 is presented on the surface of some tumor cells by the cell-surface MHC-class I molecule HLA-A1. When HLA-A1 complexes with the tumor rejection antigen of MAGE-1, the antigen is presented to a cytolytic T cell, which lyses the presenting cell. Comparisons of various MAGE genes to the region of the MAGE-1 gene coding for the nonapeptide indicated that there are homologous sequences in other MAGE genes (U.S. Patent No. 5,851,523). Thus, a family of MAGE-derived nonapeptides having the same N- and C-terminal amino acids has been identified.

Nonapeptides are of sufficient size to constitute an antigenic epitope. Antibodies generated to such nonapeptides can identify the nonapeptide alone or as part of a larger polypeptide. Like MAGE- 1 , MAGE-3 has been shown to complex with HLA-A1. MAGE-

3 also has been shown to complex with HLA-A2 (U.S. Patent No. 5,851,523).

While it has been appreciated that MAGE-12 is significantly expressed in a number of tumors of different histological type (dePlaen et al., Immunogenetics 40: 360-369 (1994), supra) and may produce tumor rejection antigens (dePlaen et al. (1994), supra; deSmet et al., Immunogenetics 39: 121-129 (1994)), the determination of the DNA and amino acid sequences of MAGE-12 (NCBI database accession 154519; GenBank database accession LI 8877; dePlaen et al. (994), supra) and sequence alignments of the regions containing the largest open reading frame in the last exons of MAGE-1, -2, -3, -4a, -6 and -12 (dePlaen et al. (1994), supra) have not led to the identification of a tumor rejection antigen in MAGE-12. It is an object of the present invention to provide such an antigen. This and other objects and advantages, as well as additional inventive features, of the present invention will become apparent to one of ordinary skill in the art from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention provides an isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL (SEQ ID NO: 1). The present invention further provides an isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding the amino acid sequence VRIGHLYIL, such as SEQ ID NO: 2. Also provided is a vector comprising and expressing such a nucleic acid molecule and a cell comprising such a vector. A composition comprising the polypeptide in an amount effective to induce an immune response to the polypeptide in a mammal as well as a composition comprising the vector in an amount sufficient to express an immune response-inducing amount of the polypeptide VRIGHLYIL in a mammal are also provided. In addition, a method of inducing an immune response to a cancer in a mammal having cancer, such as melanoma, is provided. The method comprises administering to the mammal the composition comprising the polypeptide or the vector, whereupon an immune response to the cancer in the mammal is induced. Preferably, the mammal has HLA-Cw*0702. The method can further comprise administering a cytokine, such as interleukin-2.

BRIEF DESCRIPTION OF THE FIGURE Fig. 1 represents the nucleotide (line a; SEQ ID NO: 3) and deduced amino acid (line c; SEQ ID NO: 4) sequences of MAGE-12 as compared to the nucleotide (line b; SEQ ID NO: 5) and deduced amino acid (line d; SEQ ID NO: 6) sequences of MAGE-12 published in GenBank.

DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated, at least in part, on the surprising and unexpected discovery of a T-cell epitope in the cancer-specific antigen MAGE-12 at amino acids 170-178. The epitope is recognized by tumor-infiltrating lymphocytes. The epitope is advantageous in that it provides a tumor rejection antigen for a large segment i.e., approximately 20%, of the population afflicted with melanoma, which expresses the cell-surface MHC-class I molecule HLA-Cw*0702 and for which no tumor rejection antigen is currently available for induction of an immune response to cancer. While the epitope is particularly useful in the treatment of melanoma, given that MAGE-12 is found on most tumors, the epitope has widespread utility in cancer immunotherapy.

In view of the above, the present invention provides an isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL (SEQ ID NO: 1). The polypeptide can contain up to a few, such as one, two or three, additional amino acids, for example, at either or both ends as long as the additional amino acids do not interfere with the ability of the polypeptide to function in accordance with the present invention. In addition, the polypeptide can comprise one or more variant amino acids, which desirably do not eliminate the ability of the polypeptide to function in accordance with the present invention and preferably improve the ability of the polypeptide to function in accordance with the present invention. The generation of variant polypeptides is within the ordinary skill in the art (see, e.g.,

Parkhurst et al., J. Immunol. 157: 2539-2548 (1996)). This polypeptide can be easily synthesized by Merrifield synthesis or other pep tide synthesis methodologies. GMP grade polypeptide is produced by solid-phase synthesis techniques by Multiple Peptide Systems, San Diego, CA. The polypeptide also can be recombinantly produced, if so desired, in accordance with methods known in the art.

The polypeptide complexes with cell-surface MHC-class I HLA-Cw*0702 molecules. Thus, the polypeptide can be used to identify whether or not HLA- Cw* 0702 -presenting cells are present in a sample. For example, the polypeptide can be contacted with a cell sample and the binding of HLA-Cw*0702 -presenting cells to the polypeptide can be determined using detection methods, such as radiolabeling or binding to a solid phase, known in the art.

The polypeptides and complexes thereof with HLA-Cw*0702 can be used in the generation of monoclonal antibodies or cytolytic T-cell clones specific for the polypeptides or complexes. The production of antibodies is within the skill in the art as is the generation of a cytolytic T-cell clone (see, e.g., U.S. Patent No. 5,851,523). Such monoclonal antibodies and cytolytic T-cell clones are useful reagents in diagnosing cancer; etc. Diagnostic assays, such as immunoassays and the chromium release assay, are known in the art. Cytolytic T-cell clones are also useful in adoptive transfer and other therapeutic applications (see, e.g., U.S. Patent No. 5,558,995). The present invention further provides an isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding the amino acid sequence VRIGHLYIL. Such a nucleic acid molecule can be synthesized in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence. An example of a preferred nucleotide sequence is that of SEQ ID NO: 2 (see nucleotide sequence in line "a" of Figure 1 corresponding to underlined amino acid sequence (which is SEQ ID NO: 1) in line "c" of Figure 1). Thus, the present invention also further provides a vector comprising and expressing an above-described isolated and purified nucleic acid molecule. Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements. The vector utilized in the context of the present invention desirably comprises sequences appropriate for introduction into cells. For instance, the vector can comprise an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cts-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.

Thus, according to the invention, a "vector" encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains one or more heterologous or recombinant DNA sequences, e.g., an above-described isolated and purified nucleic acid molecule, under the control of a functional promoter and, possibly, also an enhancer, and that is capable of functioning as a vector as that term is understood by those of ordinary skill in the art. Appropriate phage and viral vectors include, but are not limited to, lambda (λ) bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus.

Reference to a vector or DNA sequences contained therein as "recombinant" merely acknowledges the linkage of DNA sequences that are not typically conjoined as isolated from nature. A "gene" is any nucleic acid sequence coding for a polypeptide or protein or a nascent niRNA molecule. Whereas a gene comprises coding sequences plus any noncoding (e.g., regulatory) sequences, a "coding sequence" does not include any noncoding DNA. In the context of the present invention, a coding sequence encodes the amino acid sequence VRIGHLYIL (SEQ ID NO: 1) and consists essentially of a nucelotide sequence encoding the amino acid sequence of SEQ ID NO: 1, such as the nucleotide sequence of SEQ ID NO: 2. A "promoter" is a DNA sequence that directs the binding of RNA polymerase and, thereby, promotes RNA synthesis. "Enhancers" are -acting elements of DNA that stimulate or inhibit transcription of adjacent genes. An enhancer that inhibits transcription also is termed a "silencer." Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed "promoter elements") in that enhancers can function in either orientation, and over distances of up to several kilobase (kb) pairs, even from a position downstream of a transcribed region.

Preferably, a vector according to the invention is compatible with the cell into which it is introduced, e.g., is capable of imparting expression on the cell of the coding sequence, and is stably maintained or relatively stably maintained in the host cell. Desirably, the vector comprises an origin of replication functional in the cell. When a coding sequence is transferred, optimally the vector also contains a promoter that can drive expression of the coding sequence and that is operably linked to the coding sequence. A coding sequence is "operably linked" to a promoter gene when the promoter can direct transcription of the coding sequence. In a recombinant vector of the present invention, preferably all the proper transcription (e.g., initiation and termination signals), translation (e.g., ribosome entry or binding site and the like) and processing signals (e.g., splice donor or acceptor sites, if necessary, and polyadenylation signals) are arranged correctly on the vector, such that the coding sequence will be appropriately transcribed and translated in the cells into which it is introduced. The manipulation of such signals to ensure appropriate expression in host cells is well within the knowledge and expertise of the ordinarily skilled artisan. A native promoter or another promoter, including a constitutive promoter, such as, for instance the adenoviral type 2 (Ad2) or type 5 (Ad5) major late promoter (MLP) and tripartite leader, the cytomegalovirus (CMV) immediate early promoter/enhancer, the Rous sarcoma virus long terminal repeat (RSV-LTR), and others, including promoters appropriate for expression in prokaryotic cells, can be employed to command expression of the coding sequence.

Alternately, a tissue-specific promoter (i.e., a promoter that is preferentially activated in a given tissue and results in expression of a gene product in the tissue where activated) can be used in the vector when employed for expression in an animal host, or in cells, tissues or organs of the host. Such promoters include, but are not limited to, the elastase I gene control region, which is active in pancreatic acinar cells as described by

Swift et al. (Cell. 38, 639-646 (1984)) and MacDonald (Hepatology. 7, 425-515 (1987)). Similarly, a promoter that is selectively activated at a particular developmental stage can be employed, e.g., globin genes are transcribed differentially in embryos and adults.

Another option is to use an inducible promoter, such as the IL-8 promoter, which is responsive to TNF, or to use other similar promoters responsive to other factors present in a host or that can be administered exogenously. According to the invention, any promoter can be altered by mutagenesis, so long as it has the desired binding capability and promoter strength.

Preferably, the vector also comprises some means by which the vector or its contained subcloned sequences can be identified and selected. Vector identification and/or selection can be accomplished using a variety of approaches known to those of ordinary skill in the art. For instance, a vector comprising an above-described nucleic acid molecule can be identified by hybridization, the presence or absence of so-called "marker" gene functions encoded by marker genes present on the vectors, and/or the expression of particular sequences. In the first approach, the presence of a particular sequence in a vector can be detected by hybridization (e.g., by DNA-DNA hybridization) using probes comprising sequences that are homologous to the relevant sequence. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain marker gene functions, such as resistance to antibiotics, thymidine kinase activity, and the like, caused by particular genes encoding these functions present on the vector. In the third approach, vectors can be identified by assaying for a particular gene product, i.e., the T-cell epitope of MAGE- 12 (amino acids 170-178), encoded by the vector. Such assays can be based on the physical, immuno logical, or functional properties of the gene product.

Thus, the present invention further provides a cell comprising an above- described vector. A vector according to the present invention can be introduced into any suitable host cell, whether eukaryotic or prokaryotic. Suitable prokaryotic host cells include, but are not limited to, Escherichia coli, Bacillis subtilis, Pseudomonas aeruginosa, and members of the genus Salmonella (e.g., S. typhimurium, S. typhi, S. enteritidis, and the like). Preferably, a prokaryotic host cell is avirulent. Suitable eukaryotic host cells include, but are not limited to, rodent or mouse cells, Saccharomyces cerevisiae, and, particularly, human cells. Preferably, the vector comprises an expression vector appropriate for expression of a the coding sequence in a human or rat cell, or, alternately, in an E. coli cell. The isolation of such cells, and/or the maintenance of such cells or cell lines derived therefrom in culture, has become a routine matter, and one in which the ordinarily skilled artisan is well versed.

The form of the introduced vector can vary with the rationale underlying the introduction of the vector into the host cell. For example, the nucleic acid can be closed circular, nicked, or linearized, depending on whether the vector is to be maintained extragenomically (i.e., as an autonomously replicating vector), integrated as a provirus or prophage, transiently transfected, transiently infected as with use of a replication- deficient or conditionally replicating virus or phage, or stably introduced into the host genome through double or single crossover recombination events.

Any appropriate means of introducing the vector into a host cell can be employed. In the case of prokaryotic cells, vector introduction can be accomplished, for instance, by electroporation, transformation, transduction, conjugation or triparental mating. For eukaryotic cells, vectors can be introduced through the use of, for example, electroporation, transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate- DNA precipitate, direct microinjection into single cells, and the like. Other methods are available and are known to those skilled in the art.

A composition comprising the above-described polypeptide in an amount effective to induce an immune response (as described herein below) in a mammal to said polypeptide is further provided by the present invention. Similarly, a composition comprising an above-described vector in an amount sufficient to express an immune response-inducing amount (as described herein below) of the polypeptide VRIGHLYIL in a mammal is provided. Desirably, the composition comprises an excipient, such as a vehicle, adjuvant, carrier or diluent, which is desirably pharmaceutically acceptable, as are known in the art. See, for example, Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, eds. (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4^th ed. (1986).

A composition suitable for use in the present inventive method comprises 1 mg of the polypeptide consisting essentially of the amino acid sequence VRIGHLYIL and Montanide ISA-51 (Seppic, Inc., Fairfield, NJ). The composition is an emulsion that is suitable for injection. The composition preferably is divided into two equal volumes (1 ml each), each of which is injected into the anterior thigh deep subcutaneous tissue within 2 cm of each other. Alternatively, the polypeptide can be complex ed with liposomes. The composition can further comprise the use of an immunoadjuvant, such as incomplete Freund's adjuvant.

Thus, the present invention further provides a method of inducing an immune response to a cancer in a mammal having the cancer. In one embodiment, the method comprises administering to the mammal a composition comprising the above- described polypeptide in an amount effective to induce an immune response to the polypeptide in the mammal. In another embodiment, the method comprises administering to the mammal a composition comprising an above-described vector in an amount sufficient to express an immune response-inducing effective amount of the polypeptide VRIGHLYIL in the mammal. Any cancer that is characterized by the presentation of the T-cell epitope VRIGHLYIL, such as by the cell-surface MHC class I molecule HLA-CW*0703, can be treated in accordance with this method. Preferably, the cancer is melanoma, such as cutaneous melanoma or ocular melanoma. Non-small cell lung carcinoma, head and neck tumors, bladder carcinoma, esophageal carcinoma, sarcoma, and mammary and colorectal carcinomas are non- limiting examples of other cancers that can be treated in accordance with the present inventive method. In the context of the present inventive method, the mammal preferably has HLA-Cw*0702.

One skilled in the art will appreciate that various routes of administering a composition in the context of the present inventive method are available, and, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route.

Accordingly, there are a wide variety of suitable formulations of compositions that can be used in the present inventive methods. The means of administration is determined, in part, by whether a polypeptide or a vector is being administered. Suitable routes of administration include peritumoral, intratumoral, intravenous, intramuscular, intraperitoneal, intraurethral, subcutaneous, oral, rectal, intranasal, canulation of the ductal epithelium of the mammary gland, and the like. Peritumoral and intratumoral routes of administration, such as by injection, are preferred. Administration by lipofection, direct DNA injection, microprojectile bombardment, liposomes, molecular conjugates (with respect to recombinant DNA vectors and RNA genomes, for example), and the like, also can be effected. As an alternative to in vivo administration, cells can be removed, contacted with recombinant vector and returned to the mammal in an ex vivo technique. However, the method is not dependent on any particular means of administration and is not to be so construed. Means of administration are well-known to those ordinarily skilled in the art.

The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to induce an immune response to a cancer in the mammal having the cancer over a reasonable time frame. The dose will be determined, in part, by whether a polypeptide or a vector is employed for treatment, the severity of the cancer, as well as the body weight and age of the afflicted individual.

The dosage can be in unit dosage form, such as a tablet or capsule. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a polypeptide or vector, alone or in combination with other anticancer agents, calculated in an amount sufficient to induce an immune response, and other anti- cancer effects as desired, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular embodiment employed. The dose administered should be an "immune response-inducing amount" or an "amount effective to induce an immune response" in the individual mammal.

Since the effective amount is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism. The effective amount can be defined, for example, as the blood or tissue level desired in the mammal that corresponds to a concentration of the polypeptide or vector according to the invention, which induces an immune response. The effective amount also can vary when the compositions of the present invention are used in combination with other known anti- cancer agents. One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired effective amount in the individual mammal. One skilled in the art also can readily determine and use an appropriate indicator of the effective amount of the composition of the present invention by a direct or indirect analysis of appropriate patient samples (e.g., blood and/or tissues).

Preferably, the polypeptide is administered. If the polypeptide is administered, preferably it is administered every 1-3 weeks for four cycles, wherein four cycles equals one course. Preferably, a maximum of 24 injections is administered.

If the vector is administered, generally, an amount of vector to achieve a tissue concentration of about 10 to about 10¹² particles per ml is preferred, especially of about 10⁶ to about 10¹⁰ particles per ml. In certain applications, multiple daily doses are preferred. Moreover, the number of doses will vary depending on the means of delivery and the particular vector administered.

Further, with respect to determining the effective amount in a mammal for treatment of cancer, suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy against cancer of recombinant DNA protocols (see, e.g., PCR). These models include nude mice and SCTD mice. Such models also can be used to evaluate the in vivo efficacy of an RNA genome.

The composition can contain other active agents when used to treat cancer therapeutically. In particular, it is contemplated that an anticancer agent be employed, such as, preferably, a recombinant virus, a nucleic acid/liposomal formulation (or other nucleic acid delivery formulation), or another vector system (e.g., retrovirus or adenovirus), either as a viral particle or as a nucleic acid/liposomal formulation. Further representative examples of these additional pharmaceuticals that can be used in addition to those previously described, include chemotherapeutic agents, immunostimulants, antiviral compounds, and other agents and treatment regimes (including those recognized as alternative medicine) that can be employed to treat cancer. Anticancer compounds include, but are not limited to, angiostatin, endostatin, anti-HER-2/neu antibody, and tamoxifen. Immunomodulators and immunostimulants include, but are not limited to, various interleukins, cytokines, antibody preparations, and interferons. Preferably, the method of the present invention further comprises administering a cytokine. The cytokine can be any suitable cytokine in the context of cancer immunotherapy and includes all forms of such molecules, whether human, murine or other, and whether isolated from nature or produced recombinantly, as well as any and all variants of such molecules, provided that the variants have the same activity, whether to a greater or lesser extent. Preferably, the cytokine is interleukin-2 (IL-2). Preferred amounts of a given cytokine to be administered by a given route are set forth in Biologic Therapy of Cancer, deVita, Hellman and Rosenberg, eds., J.P. Lippincott Co., Philadelphia, PA, 2^nd ed. (1995). If a cytokine, such as IL-2, preferably recombinant human IL-2 (rhTL-2; available from Chiron; desirably reconstituted and preferably further diluted with human serum albumin), is administered in the context of the present inventive method, preferably it is administered as a high-dose intravenous bolus of 720,000 IU/kg over 15 min every 8 hrs, beginning on the day after immunization and continuing for up to 4 days for a maximum of 12 doses. Doses can and should be skipped if a patient develops grade III or IV toxicity, which can be easily reversed by temporarily discontinuing administration, such that additional doses of cytokine, e.g., rhIL-2, can be subsequently administered.

While the cytokine, itself, can be administered, a vector (as described above) encoding and expressing the cytokine at levels described above can be administered. Alternatively, a universal immunomodulatory cytokine-expressing bystander cell line, such as one that expresses IL-2, can be administered (see, e.g., Levitsky et al., WO 99/38954) such that the desired level of cytokine is achieved.

The following example serves to illustrate further the present invention and is not intended to limit its scope in any way.

EXAMPLE This example describes the identification of a T-cell epitope in MAGE-12. Tumor-infiltrating lymphocyte (TIL) cell lines and a melanoma (MEL) cell line were established from a patient (F001) with metastatic melanoma in accordance with methods known in the art (Panelli et al, J Immunol. 164: 495-504 (1999)). The TIL was designated F001-TIL and the MEL was designated F001-MEL (an early passage (<5 passages) melanoma cell line; (Riker et al., Cancer Detect. Prev. 23: 387 (1999)). The cell lines were subjected to detailed functional and molecular testing. It was determined that the HLA allele Cw*0702 was the restriction element for F001- TIL. A CDNA library from F001-MEL transfected into interferon-α (IFN-α)- stimulated 293 human embryonal kidney cells expressing HLA-Cw*0702 identified a cDNA sequence homologous, except for one amino acid (amino acid 187 D → A), to MAGE-12 as the target antigen for FOOl -TIL. Based on the binding motif of HLA- Cw*0702 (Falk et al, PNAS USA 190: 12005 (1993)), a library of 25 polypeptides from the MAGE-12 sequence was synthesized and screened by culturing F001-EBV- B cells (B lymphoblastoid cells derived from FOOl and transformed with exogenous EBV (Moss et al., Nature 331 : 719 (1988)) with 10 μM and 1 μM concentrations of each peptide to map the FOOl -TIL epitope. In this fashion, four candidate peptides were identified that could stimulate IFN-γ release by F001-ΗL at 1 μM concentration. Titration of these candidate epitopes identified one polypeptide, namely amino acids 170-178 of MAGE-12, that could stimulate IFN-γ release by FOOl -TIL at 10-100 picomolar concentration. Alignment of this polypeptide with the polypeptide sequences of other MAGE proteins commonly expressed by tumor cells revealed limited homology; the closest sequences were those from MAGE-2 and MAGE-3, with two and three amino acid differences, respectively. Fig. 1 represents the nucleotide (line a) and deduced amino acid (line c) sequences of MAGE-12 derived from analysis of cDNA from FOOl -MEL. The sequences are aligned with the MAGE-12 nucleotide (line b) and deduced amino acid (line d) sequences available through GenBank (GenBank accession no. LI 8877). The nucleotides and amino acids are numbered from the first codon and amino acid, respectively. The variant residue and related nucleotide at position 187 are underlined. The epitope recognized by FOOl -TEL is underlined (amino acids 170- 178).

Thus, amino acids 170-178 of MAGE-12 represents an epitope that interacts with the MHC class I molecule HLA-Cw*0702. The resulting complex stimulates the production of cytolytic T-cell clones that lyse the presenting cells. The epitope meets a long-felt need for a tumor rejection antigen in the large segment (i.e., 20%) of patients with melanoma for which no tumor rejection was previously available.

Incorporation by Reference All sources (e.g., inventor's certificates, patent applications, patents, printed publications, repository accessions or records, utility models, World-Wide Web pages, and the like) referred to or cited anywhere in this document or in any drawing, Sequence Listing, or Statement filed concurrently herewith are hereby incorporated into and made part of this specification by such reference thereto.

Guide to Interpretation The foregoing is an integrated description of the invention as a whole, not merely of any particular element or facet thereof. The description describes "preferred embodiments" of this invention, including the best mode known to the inventors for carrying it out. Of course, upon reading the foregoing description, variations of those preferred embodiments will become obvious to those of ordinary skill in the art. The inventors expect ordinarily skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. As used in the foregoing description and in the following claims, singular indicators (e.g., "a" or "one") include the plural, unless otherwise indicated. Recitation of a range of discontinuous values is intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually listed. As regards the claims in particular, the term "consisting essentially of indicates that unlisted ingredients or steps that do not materially affect the basic and novel properties of the invention can be employed in addition to the specifically recited ingredients or steps. In contrast, the terms "comprising," "having," or "incorporating" indicate that any ingredients or steps can be present in addition to those recited. The term "consisting of indicates that only the recited ingredients or steps are present, but does not foreclose the possibility that equivalents of the ingredients or steps can substitute for those specifically recited.

Claims

WHAT IS CLAIMED IS:

1. An isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL (SEQ ID NO: 1).

2. An isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding the amino acid sequence VRIGHLYIL.

3. The isolated and purified nucleic acid molecule of claim 2, in which the nucleotide sequence encoding the amino acid sequence VRIGHLYIL is SEQ ED NO:

2.

4. A vector comprising and expressing the isolated and purified nucleic acid molecule of claim 2 or 3.

5. A cell comprising the vector of claim 4.

6. A composition comprising the polypeptide of claim 1 in an amount effective to induce an immune response in a mammal to said polypeptide.

7. A composition comprising the vector of claim 4 in an amount sufficient to express an immune response-inducing amount of the polypeptide VRIGHLYEL in a mammal.

8. A method of inducing an immune response to a cancer in a mammal having said cancer, which method comprises administering to said mammal a composition of claim 6 or 7, whereupon an immune response to said cancer in said mammal is induced.

9. The method of claim 8, wherein said cancer is melanoma.

10. The method of claim 8, wherein said mammal has HLA-Cw*0702.

11. The method of claim 8, 9 or 10, which further comprises administering a cytokine.

12. The method of claim 11, wherein said cytokine is interleukin-2.