WO2000024778A1 - Hla-a2 and hla-dr specific peptide epitopes from the melanoma antigen trp2 - Google Patents

Abstract

Novel HLA-Class I and HLA Class II restricted epitopes of the melanoma antigen tyrosinase-related protein 2 are described. A novel HLA-A*0201 restricted epitopes present in the melanoma antigen tyrosinase-related protein 2 (TRP2) is disclosed. The invention relates to a nine-amino acid peptide designated TRP2 (180-188). The peptide is demonstrated to induce cytotoxic T lymphocytes (CTL) which specifically react with, and lyse, melanoma cells in the context of HLA-A2 or HLA-A*0201. The invention further relates to a nine-amino acid peptide derived from TRP2 which induces helper T lymphocytes in the context of HLA-DR. The invention also relates to diagnostic methods and pharmaceutical compositions which employ the tumor antigen therapeutically and prophylactically.

Description

TITLE OF THE INNENTION

HLA-A2 AND HLA-DR SPECIFIC PEPTIDE EPITOPES FROM THE

MELANOMA ANTIGEN TRP2

FIELD OF THE INVENTION

The present invention relates to the area of cancer diagnostics and therapeutics. More specifically, the invention relates to the identification of novel HLA-A2 and HLA-DR specific epitopes from the melanoma antigen tyrosinase-related protein 2 (TRP2) and their use in diagnostic methods. The invention further relates to pharmaceutical compositions which employ these peptides, therapeutically and prophylactically.

BACKGROUND OF THE INVENTION Clinical evidence strongly suggests that T lymphocytes can mediate the regression of melanoma. In one study, the adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) with IL-2 significantly reduced tumor burden in 30- 40% of patients with metastatic disease (1, 2). Some populations of tumor reactive cytotoxic T lymphocytes (CTL) derived from TIL or from lymphocytes stimulated in vitro with tumor cells recognize non-mutated proteins expressed on melanomas from multiple patients. At least 14 normal self proteins have been identified as melanoma antigens (3-5), and results from several clinical investigations suggest that class I MHC restricted recognition of epitopes from such proteins by CTL may be involved in tumor regression (see, for example, 6-9).

One such self protein is tyrosinase-related protein 2 or TRP2 (see 10, 11 and WO 97/29195). TRP2 has been identified as a melanoma antigen recognized by tumor reactive CTL in both the mouse (12) and human (13). In the murine model, several B16 reactive CTL lines generated from splenocytes of C57BL/6 mice immunized with irradiated B16 melanoma cells recognized a TRP2(181-188) (VYDFFVWL: SEQ ID NO: 4) antigen in the context of H-2K^b. In addition, a T cell line raised by repeated in vitro stimulation of murine splenocytes with the TRP2 (181- 188) peptide recognized B 16 melanoma and eliminated 3 day old established pulmonary micrometastases in vivo (12). In the human, the TRP2 (197-205) (LLGPGRPYR: SEQ ID NO: 56) peptide was identified as the HLA-A31 restricted epitope recognized by a CTL clone derived from a population of TIL (TIL586). The adoptive transfer of this TIL with IL-2 into the autologous patient resulted in an objective clinical response (13). The same peptide was also recognized by an independent TIL (TIL 1244) in the context of HL A- A33 (14).

However, HLA-A31 and HLA-A33 were only expressed in 6% and 2% respectively of the 412 melanoma patients referred to the National Cancer Institute (15). By comparison, HLA-A2 is expressed in about 47% of melanoma patients in the United States with the most common subtype HLA-A^*0201 expressed in approximately 98% of the HLA-A2 positive patients in North America (16). In addition, since the expression of non-mutated melanoma antigens is heterogeneous among tumors isolated from different patients and between individual cells from single lesions, the development of immunotherapies based on as many antigens as possible may be clinically beneficial. Therefore, to increase the number of patients eligible for TRP2 based treatments, it is desirable to identify additional HLA-A2 restricted epitopes from the TRP2 protein.

A number of antigens that are recognized by human class II restricted, melanoma reactive T cells have now been described. Initial studies demonstrated that CD4⁺ T cell clones raised by the stimulation of PBL, as well as CD4⁺ tumor infiltrating lymphocytes (TIL) could recognize autologous melanoma cells (Chen, Q. and Hershey, P. 1992) (Radrizzani, M. et al 1991). Additional data indicated that CD4⁺ T cells could recognize shared melanoma antigens (Topalian et al, Int. J. Cancer. 1994). Subsequently, the widely shared melanosomal antigen tyrosinase was found to be recognized by HLA-DRB 1*0401 reactive T cells and 2 peptide epitopes from this molecule were identified (Topalian et al, J. Exp. Med. 1996). The cancer-testis antigen MAGE-3, a shared melanoma antigen identified initially through the use of class I restricted T cells, has also been shown to be recognized by class II restricted T cells. In one study, in vitro sensitization carried out with the recombinant MAGE-3 protein was shown to elicit T cells that recognized peptide epitopes from this antigen in the context of the class II HLA-DRB1*1301 and 1302 alleles (Chaux et al, 1999, L Exp. Med .

Additional studies have resulted in the identification of unique antigens recognized by class II restricted, melanoma reactive T cells. Using a biochemical approach, a mutated triosephosphate isomerase epitope was found to be recognized by HLA-DRB1*0101 restricted, melanoma reactive T cells (Pieper et al, J. Exp. Med. 1999). A novel product generated by a chromomosomal rearrangement resulted in the juxtapositon of sequences from the low density lipid receptor and the 2-a-L- fucosyltransferase gene, and a T cell epitope that was recognized in the context of HLA-DRB1*0101 was identified (Wang et al, J. Exp. Med. 1999). In addition, a mutated product of the CDC27 gene was found to be recognized by HLADRB 1*0401 restricted, melanoma reactive T cells (Wang et al, Science. 1999).

It is therefore desirable to identify other novel HLA-Class II restricted cancer associated peptide epitopes for use in immunotherapy.

SUMMARY OF THE INVENTION The invention relates to the identification of HLA-Class I and -Class II restricted epitopes present in tyrosinase-related protein 2, and analogs of the epitopes.

The invention relates to the identification of HLA-A2 restricted epitopes present in the 519 amino acid melanoma antigen known as tyrosinase-related protein 2 (TRP2). In particular, the invention relates to a nine-amino acid peptide designated TRP2 (180-188) which has the amino acid sequence SVYDFFVWL and is demonstrated to induce cytotoxic T lymphocytes (CTL) which specifically react with, and lyse, melanoma cells in the context of HLA-A*0201. The invention further relates to analogs of TRP2 (180-188) which are capable of specifically reacting with, and lysing, melanoma cells in the context of HLA- A*0201.

The invention therefore also relates to nucleic acid sequences which encode TRP2 (180-188) and analogs thereof. The invention further relates to a diagnostic method which utilizes TRP2 (180-188) and/or analogs thereof to detect melanoma in a mammal.

The invention also relates to pharmaceutical compositions which comprise TRP2 (180-188), and/or analogs thereof, either alone or in combination with other HLA-A2 specific peptides encoded by TRP2 or other melanoma antigens, and the use of these compositions in the prevention or treatment of melanoma in a mammal.

The invention further relates to pharmaceutical compositions which comprise nucleic acid molecules encoding TRP2 (180-188), and or analogs thereof, either alone or in combination with nucleic acid sequences encoding other HLA-A2 specific melanoma antigen peptides and the use of these compositions in the prevention or treatment of melanoma in a mammal.

The invention therefore also relates to methods of producing a TRP2 (180-188)-specifιc T cell response in a mammal utilizing the compositions of the invention. The present invention further relates to isolated T cells having specific reactivity to the TRP2 (180-188) peptide or analogs thereof, to methods of preparing such T-cells, to the use of such T cells as diagnostics and therapeutic reagents, and to pharmaceutical compositions comprising the T cells.

The invention also relates to target cells, preferably dendritic cells, which have been incubated in vitro with the TRP2 (180-188) peptide and or analogs thereof, to the use of such target cells as diagnostic and therapeutic reagents, and to pharmaceutical compositions which comprise the target cells.

The invention relates to TRP2 peptides having at least 9 amino acids and derived from TRP2 which comprise the amino acid sequence Xaa, LPYWNFAT Xaa^, wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaaj is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II. The invention further relates to nucleic acid sequences which encode a TRP2 peptide comprising at least 9 amino acids comprising the amino acid sequence Xaa, LPNWNFAT Xaaj and analogs thereof, wherein Xaa, any one of 20 naturally occurring amino acids, preferably is an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa_j is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II.

Another aspect of the invention is a vector comprising nucleic acid sequences which encode a TRP2 peptide comprising at least 9 amino acids and comprising the amino acid sequence Xaa, LPYWNFATXaa,, and analogs thereof, wherein Xaa, any one of 20 naturally occurring amino acids, preferably is an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaaj is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II.

Yet another aspect of the invention are host cells transformed or transfected by the vector encoding a TRP2 peptide and optionally are transformed or transfected with an HLA-Class II molecule and the use of the host cells as immunogens or vaccines against cancer. The invention further provides a diagnostic kit and a diagnostic method which utilizes the TRP2 peptides having at least 9 amino acids comprising the amino acid sequence Xaa, LPYWNFATXaa;, wherein Xaa, any one of 20 naturally occurring amino acids, preferably is an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa_j is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II or analogs thereof, or the nucleic acid sequence encoding same, or complementary nucleic acid sequence to detect melanoma in a mammal.

Another aspect of the invention is a pharmaceutical composition comprising a TRP2 peptide having at least 9 amino acids and comprising the amino acid Xaa,LPYWNFATXaa₂, wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa_j is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II, or analogs thereof, or combinations thereof, either alone or in combination with other HLA-Class II specific peptides encoded by TRP 2 or other melanoma antigens for use as an immunogen for eliciting specific helper T cell immune responses and as a vaccine in the prevention or treatment of melanoma in a mammal. The invention further relates to pharmaceutical compositions which comprise nucleic acid sequences encoding a TRP2 peptide comprising at least 9 amino acids and comprising the amino acid sequence Xaa, LPYWNFATXaa^ wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa_j is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class H, or combinations thereof, alone or in combination with nucleic acid sequences encoding other HLA Class Il-specific melanoma antigen peptides for use in the prevention or treatment of melanoma in a mammal. The invention further relates to methods of producing an HLA-Class II restricted T cell response to a TRP2 peptide having at least 9 amino acids and comprising the amino acid sequence Xaa,LPYWNFATXaa₂ wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and XaO is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II, in a mammal utilizing the compositions of the present invention.

The present invention further relates to isolated T cells having specific reactivity to a TRP2 peptide having at least 9 amino acids and comprising the amino acid sequence Xaa,LPYWNFATXaa₂ wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa, is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II to methods of preparing the specific T cells, and the use of the T cells as a diagnostic and therapeutic use.

Another aspect of the invention are antigen presenting cells (APC) expressing an appropriate MHC Class II molecule pulsed or transfected with the TRP2 peptide having at least 9 amino acids and comprising the amino acid sequence Xaa,LPYWNFATXaa₂ wherein Xaa, any one of 20 naturally occurring amino acids, preferably is an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaaj is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof which are recognized by immune cells in the context of HLA Class II or analogs thereof for use as a diagnostic or therapeutic reagent. Another obj ect of the invention is to provide a method of monitoring the efficacy of a cancer vaccine therapy in a mammal comprising (A) isolating T lymphocytes from the vaccine-treated mammal (B) measuring immunoreactivity of the T lymphocytes in the presence of the TRP2 peptide having at least 9 amino acids and comprising the amino acid sequence Xaa,LPYWNFATXaa₂ wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa, is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof, an enhancement of immunoreactivity in comparison to immunoreactivity of control lymphocytes is indicative of efficacy. The present invention also relates to polyclonal, monoclonal and recombinant antibody elicited by and immunoreactive with, the TRP2 peptide having at least 9 amino acids and comprising the amino acid sequence Xaa,LPYWNFATXaa₂ wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid and Xaa_j is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid, and analogs thereof for use as a diagnostic and/or therapeutic reagent.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 A through IF show the lysis of TRP2 (180-188) peptide pulsed target cells by bulk CTL from patients following 6 in vitro restimulations of the CTL with peptide-pulsed target cells. Specific lysis of control T2 cells(i.e. T2 cells not pulsed with peptide) was compared to that of T2 cells pulsed with 1 M TRP2 (180-188) by CTL from patients AN (a), MU (b), and KU (c) in 4 hr ⁵¹Cr release cytotoxicity assays. In addition, specific lysis of 3 HLA-A2⁺TRP2⁺ melanoma cell lines was compared to that of 2 HLA-A2^'TRP2⁺ and 1 HLA-A2⁺TRP2^" melanoma lines by bulk CTL from patients AN (d), MU (e), and KU (f).

DETAILED DESCRIPTION OF THE INVENTION The invention relates to the identification of HLA Class I or HLA Class II restricted peptides from the melanoma antigen, tyrosinase-related protein 2.

The invention relates to the identification of an HLA-A2 restricted peptide epitope, from the melanoma antigen tyrosinase-related protein 2 (TRP2). This peptide, designated TRP2 (180-188), was identified by screening TRP2 derived peptides for the ability to induce cytotoxic T lymphocytes (CTL) which specifically react with, and lyse, melanoma cells in the context of HLA-A*0201.

The invention therefore relates to the amino acid sequence of TRP2 (180- 188), which is shown in SEQ ID NO: 1, or analogs thereof where the term analog as used throughout the specification and claims in context of HLA Class I molecules refers to sequences which are biologically equivalent to the native TRP2 (180-188) peptide in that they are able to induce cytotoxic T lymphocytes (CTL) which specifically react with, and lyse, melanoma cells in the context of HLA- A^* 0201.

Since human HLA-A2 molecules recognize peptides of nine or ten amino acids in length, TRP2 (180-189) (SVYDFFVWLH, shown as SEQ ID NO: 3) constitutes an analog of TRP2 (180-188). In addition, analogs of TRP2 (180-188) include sequences in which one or more residues of SEQ ID NO:l have been conservatively substituted such that the analog is biologically equivalent to the native TRP2 (180-188) peptide. Examples of conservative substitutions include the substitution of one-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 for another. The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting peptide is biologically equivalent to the native TRP2 (180-188) peptide. Preferred positions to be conservatively substituted in TRP2 (180-188) (SEQ ID NO:l) or its analog TRP2 (180-189) (SEQ ID NO: 3) include, but are not limited to, position 1 (residue 180), position 2 (residue 181) and the carboxy-terminal position (residue 188 in TRP2 (180-188) and residue 189 in TRP2 (180-189) ).

Preferred analogs of TRP2 (180-188) include, but are not limited to, substitution of either leucine or methionine at position 2 and the substitution of leucine for valine at the carboxy terminus.

The present invention further relates to the identification of an HLA Class II restricted peptide epitope from the melanoma antigen tyrosinase-related protein 2 and the equivalent peptide epitope from TRP-1. The HLA Class II restricted TRP2 peptides of the present invention are HLA-DR restricted, preferably HLA- DRB 1*1501 or 1502 restricted. In one embodiment of the invention, the HLA Class II restricted TRP2 peptides of the present invention are expressed on melanomas derived from the majority of individuals that express the major histocompatibility complex

(MHC) Class II allele HLA-DRB1*1501 or 1502, which is expressed in the Caucasian population at a frequency of between 9 an 19%. The HLA Class II restricted TRP2 peptides and analogs of the present invention are identified by the ability to induce helper T lymphocytes which specifically react with the peptide in the context of peptide pulsed antigen presenting cells expressing the appropriate HLA-Class II molecule.

The invention provides a HLA Class Il-restricted peptide of TRP2 comprising an amino acid sequence of at least about nine amino acids in length. The HLA Class Il-restricted peptides of the present invention are as long as about 32 amino acids, preferably less than about 22 amino acids in length, more preferably between about 9 amino acids and about 16 amino acids in length. In one embodiment the peptide comprises the amino acid sequence Xaa,LPYWNFATXaa₂ (SEQ. ID NO: 60) or analogs thereof, wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acid; and Xaaj is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid.

The HLA Class II restricted TRP2 peptides of the present invention of the general formula, Xaa, LPYWNFATXaa;, may variably comprise one to about 11 additional amino acids at the N-terminus and/or one to about 11 additional amino acids at the C-terminus. In this embodiment, the HLA Class II restricted TRP2 peptide is represented by the formula, Xaa₃Xaa,LPYWNFATXaa₂Xaa₄ (SEQ ID NO: 71) wherein Xaa₃ comprises from zero to about 11 amino acids in length, preferably from zero to about 16 amino acids; and Xaa₄ comprises variably from zero to about 11 amino acids in length, preferably from zero to about 16 amino acids in length. The amino acids for Xaa₃ and Xaa₄ may be any of the 20 naturally occurring amino acids. In one embodiment Xaa₃ comprises the amino acid sequence Asp Leu Gin Arg Leu He Gly Asn Glu Ser Phe (SEQ ID NO: 72).

In one embodiment Xaa₄ comprises Arg. In yet another embodiment Xaa₄ comprises Arg Asn Glu Cys Asp Val Cys Thr Asp Gin Leu (SEQ ID NO: 73). Other embodiments of the HLA Class II restricted TRP2 peptides invention comprise LPYWNFATG (SEQ. ID NO: 61); ALPYWNFAT (SEQ. ID NO: 62); ALPYWNFATG (SEQ. ID NO: 63); SLPYWNFATG (SEQ. ID NO: 64); QLPYWNFATG (SEQ. ID NO: 65); VLPYWNFATG (SEQ. ID NO: 66); ALPYWNFATGR (SEQ. ID NO: 67); FALPYWNFATG (SEQ. IF NO: 68); LQRLIGNESFALPYWNFATG (SEQ. ID NO: 69); ALPYWNFATGRNECDVCTDQ (SEQ. ID NO: 70) and analogs of each sequence.

Analogs of the HLA-Class II restricted TRP2 peptides of the present invention are those peptides having one or more substitutions in the consensus binding motif which results in equivalent or enhanced immuno logical responses as compared to the native motif.

In one embodiment, an analog of the sequence ALPYWNFAT comprises a single substitution of Phe or He in place of Tyr or a single substitution of He, Leu, Val, or Met in place of Phe, or a combination of substitutions at each position.

In another embodiment, an analog of the sequence ALPYWNFAT comprises a single substiution of Phe, Tyr, or He in place of Tip or a single substitution of He, Leu, Val, Met, or Phe, in place of Ala, or combinations of substitutions at each position. The HLA Class I restricted TRP2 peptides or analogs thereof and HLA

Class II restricted TRP2 peptides or analogs thereof can be synthesized by automated instruments sold by a variety of manufactures or can be commercially custom-ordered and prepared. Alternatively, the peptide can be expressed from nucleic acid sequences which are capable of directing synthesis of the peptide using recombinant DNA methods known to those of ordinary skill in the art, and purified by methods known in the arts.

The present invention also encompasses a nucleic acid sequence encoding a HLA-Class I restricted peptide derived from TRP-2 or encoding an HLA- Class II restricted peptide derived from TRP-2. The nucleic acid sequence of the entire native TRP2 gene is disclosed in U.S. Patent No. 5,831,016 incorporated herein by reference.

The invention therefore relates to the nucleic acid sequence encoding TRP2 (180-188) and its analogs with a preferred nucleic acid sequence shown as SEQ ID NO: 2 (AGTGTTTATGATTTTTTTGTGTGGCTC). The invention further relates to the nucleic acid sequence encoding an HLA-Class II restricted TRP2 peptide. In one embodiment the nucleic acid sequence encodes a peptide comprising Xaa, LPYWNFATXaa, or analogs thereof, wherein Xaa, is any one of 20 naturally occurring amino acids, preferably an amino acid selected from the group consisting of Ala, Gin, Val, Ser, Leu, He or no amino acd; and Xaaj is any one of 20 naturally occurring amino acids, preferably Gly, or no amino acid. It should be noted that the nucleic acid described herein represent a preferred embodiment of the invention. Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a sequence capable of directing production of the HLA Class I restricted TRP2 (180-188) peptide or .analogs thereof and the HLA Class II restricted TPR2 peptide or analog thereof. As such, nucleic acid sequences which are functionally equivalent to the sequences described herein are intended to be encompassed within the present invention. The invention further relates to expression vectors comprising the nucleic acid sequences encoding the HLA Class I restricted TRP2 (180-188) or analogs thereof or comprising the nucleic acid sequences encoding the HLA-Class II restricted TRP2 peptide or analogs thereof. Any expression vector that is capable of carrying and expressing the nucleic acid sequences encoding the HLA Class I restricted TRP2 (180- 188) or the HLA Class II restricted TRP2 peptides or analogs thereof in prokaryotic or eukaryotic host cells may be used including but not limited to recombinant virus such as vaccinia, fowlpox or adenovirus and the like. The invention also encompasses host cells transformed, transfected or infected with the vector to express the HLA-Class II restricted TRP2 peptide or analog thereof. The host cell may endogenously express an appropriate HLA-Class II molecule or may be recombinantly engineered to express an exogenous HLA Class II molecule, using methods known in the art. As HLA-A2 is the most commonly expressed family of class I MHC molecules in melanoma patients in the United States with an estimated frequency of 47% and HLA-A^*0201 is the most common subtype, being expressed in approximately 98% of the HLA-A2 positive patients in North America. The MHC Class II allele HLA-DRB1*1501 and 1502 is expressed in the Caucasian population at a frequency of between 9 and 19%. As such, the HLA Class I and HLA Class II restricted TRP2 peptides may be used to screen for melanomas. Therefore, the present invention also relates to the use of the HLA-Class I restricted TRP2 (180-188) peptide and/or analogs thereof and use of HLA-Class II restricted TRP2 peptides in a method for detecting the presence of melanoma in a mammal, where, as used in this application, the term melanoma includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. In one embodiment, the method of detecting melanoma involves isolating cytolytic T lymphocytes (CTLs) from peripheral blood, lymph nodes or spleens of a mammal using methods known to those skilled in the art and, coincubating the CTLs in vitro with target cells pre-exposed to either TRP2 (180-188) peptide and/or analogs thereof, or to expression vector containing the nucleic acid sequences encoding the TRP2 (180-188) peptide or analogs thereof. Ratios of CTL to target cells to be used in the method range from about 1:1 to about 100:1.

Target cells to be used in the method include, but are not limited to, HLA-A2⁺TRP2⁺ melanoma cell lines, HLA-A^*0201⁺ T2 cells pulsed with TRP2 (180- 188) and or analogs thereof, or antigen presenting cells such as B cells, dendritic cells, macrophages, Langerhan cells pulsed with TRP2 (180-188) and/or analogs thereof as defined above. Target cells also include but are not limited to HLA-DRB1*1501⁺ or 1502⁺ B cells, melanoma cells, antigen presenting cells and the like pulsed with a HLA Class II restricted TRP2 peptide or analog thereof or recombinantly expressing the HLA- Class II restricted TRP peptide or analog thereof. The helper T cell response or CTL response to the target cells can be determined by a variety of methods including measuring cytokine release by the helper T cells or CTLs following coincubation with the target cells or by measuring lysis of the target cells by the CTLs.

Where cytokine release by the helper T cells or CTLs is to be utilized as an indicator of the production of a specific helper T cell or specific CTL response to the TRP2 peptides or analogs thereof, the cytokine release can be measured by methods known to those skill in the art including immunoassays such as ELISAs and radioimmunoassays.

A preferred cytokine to assay for release from stimulated CTLs is IFN as described in the Examples. Other cytokines which can be measured include, but are not limited to, GM-CSF, TNF and IL-2.

Where the lytic activity of the CTLs is to be measured as an indicator of a TRP2 (180-188) peptide specific CTL response following coincubation with target cells, the lysis of target cells can be measured using methods known to those skilled in the art including the ⁵¹Cr-release assay described in Example 2.

To determine whether the patient has melanoma, cytokine release by the CTLs or specific lysis of target cells by CTLs as measured above is then compared to the cytokine secretion or lysis of target cells by CTLs coincubated in vitro with target control cells where control cells include, but are not limited to, HLA-A2^"TRP2⁺ or HLA-A2⁺TRP2^" cells, or T2 cells or dendritic cells not exposed to peptide. A two-fold or more increase in cytokine release or cytolytic activity as compared to that of the control cells indicates the presence of melanoma.

The present invention also relates to methods of treating a mammal having melanoma. In one embodiment, the method comprises exposing T lymphocytes, preferably autologous cytotoxic lymphocytes or tumor infiltrating lymphocytes obtained from a patient with melanoma, in vitro to a TRP2 peptide and/or analogs thereof, either alone or in combination with other HLA-A2-restricted epitopes from TRP 2, or in combination with other HLA-DR restricted epitopes from TRP2, or other melanoma antigens to elicit TRP2 specific CTLs or helper T lymphocytes, and administering the TRP2 specific CTLs or helper T lymphcytes to the mammal.

It is believed that since HLA- A* 0201 restricted epitopes have been identified to date from 6 known melanoma antigens: MART-1 (17), gplOO (6, 7, 18- 20), tyrosinase (21), MAGE-3 (22, 23), N-acetylglucosaminyl-transferase-V (GnT-V) (24), and the melanocyte-stimulating hormone receptor MC1R (4) (the epitopes disclosed in references 4, 6, 7 and 17-24 are hereby incorporated by reference), immunotherapies utilizing HLA-A*0201 specific antigens are applicable to a large number of patients.

Techniques for sensitizing human T lymphocytes in vitro to tumor antigen immunodominant peptides are known in the art. In addition, lymphocytes can be subjected to repetitive in vitro stimulation to produce peptide-specific lymphocytes having a greater capacity to recognize human tumor antigens.

In another embodiment, the method of treating a mammal having melanoma comprises immunizing the mammal with the HLA Class I restricted TRP2 peptide and/or analogs thereof in an amount effective to elicit a HLA Class I restricted TRP2 (180-188) peptide-specific response. In one embodiment, the TRP2 (180-188) peptide and/or analogs thereof can be fused with an endoplasmic reticulum signal peptide as described in U. S. patent 5,733,548, hereby incorporated by reference, and the resulting chimeric protein administered to a mammal in an amount effective to prevent melanoma. In an embodiment, the method of treating a mammal having a melanoma comprises immunizing the mammal with a HLA-Class II restricted TRP2 peptide, analog, or combination thereof in an amount effective to elicit a HLA-Class II restricted TRP2 peptide specific response. The method may further provide cytokines such as IL- 2, IL-4, and the like, or adjuvants such as RLBI-Detox™, alum, and the like for enhancement of the immune response. The HLA-Class II restricted TRP2 peptides of the present invention may also be provided in the form of a TRP2 peptide-protein carrier conjugate, for enhancing the immune response. Protein carriers which may be used include but are not limited to Pseudomonas exotoxin, poly-L-lysine and the like as are known in the art. In an alternative embodiment, an expression vector containing the nucleic acid sequences encoding the TRP2 peptide or analogs thereof may be administered to the mammal having melanoma in an amount effective to elicit a TRP2 peptide-specific response. Of course, one of skill in the art would recognize that the TRP2 peptide or nucleic acid sequences encoding the peptide could be administered to the mammal in combination with other HLA-A2 restricted epitopes or other HLA-Class II restricted epitopes from TRP2 or melanoma antigen such as those identified in MART-1, gplOO, tyrosinase, MAGE-3, GnT-V and MC1R as disclosed above.

CTL populations or helper T lymphocyte populations reactive against the TRP2 peptide may then be isolated from a peripheral blood sample or spleen cells of the mammal immunized with the peptide or expression construct from about 3 to about 30 days after immunization. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas.

CTLs or helper T lymphocytes are cultured for about 7 to about 90 days (3) and then screened to determine the clones of the desired reactivity against the TRP2 peptide using known methods of assaying T cell reactivity; CTLs or helper T lymphocytes producing the desired reactivity are thus selected.

The selected CTLs or helper T lymphocytes may be administered via one of several routes including but not limited to intravenous, intraperitoneal, intramuscular or subcutaneous. A preferred route of administration is intravenously.

In general, it is desirable to provide the recipient with a dosage of about 10⁷ to about 10¹¹ CTLs or helper T lymphocytes. A preferred dosage is about 5xl0⁹ to about 5x10¹⁰ lymphocytes.

The invention further relates to CTLs or helper T lymphocytes having specific reactivity to the TRP2 peptide, and to the use of such CTLs or helper T lymphocytes as diagnostic and therapeutic agents. For example, TRP2 (180-188) specific CTLs can be used diagnostically to screen target cells (i.e. antigen presenting cells such as dendritic cells, macrophages and Langerhan cells) from a patient by measuring lysis of target cells following coincubation with the peptide-specific CTLs, wherein lysis of the target cells indicates that the patient has melanoma.

Alternatively, TRP2 specific CTLs or helper T lymphocytes can be used prognostically to detect HLA-A2⁺TRP2⁺ target cells or HLA-DRB1*1501⁺ or 1502⁺ TRP2⁺ target cells. If such target cell were isolated from a patient's tumor and were recognized by the TRP2 specific CTLs or helper T lymphocytes, the indication would be that the tumor was a melanoma. In yet another embodiment, the method of treating a mammal having melanoma comprises administering target cells which have been exposed in vitro to the TRP2 (180-188) peptide and or analogs thereof or the MHC Class II restricted TRP2 peptide or analogs thereof to a mammal in an amount effective to elicit a specific T lymphocyte response to the TRP2 peptides. For example, dendritic cells cultured for about 1 week in vitro with about 1000 u ml GM-CSF and 1000 u/ml IL-4 and pulsed with the TRP2 peptide and/or analogs thereof can be administered intravenously to a mammal at a dosage of about lxl 0⁸ to about 2xl0⁸ cells.

The invention therefore also relates to target cells which have been exposed in vitro to the TRP2 (180-188) peptide and/or analogs thereof or the MHC Class II restricted TRP2 peptide or analogs thereof. These target cells can be used as diagnostic and therapeutic reagents. For example, target cells which have been incubated in vitro with the TRP2 (180-188) peptide and/or analogs thereof can be used diagnostically to screen CTLs from a patient by measuring lysis of the target cells following incubation with the CTLs, wherein lysis of the target cells indicates that the patient has melanoma. Or in the case of an MHC Class II restricted TRP2 peptide or analog thereof, target cells which have been incubated in vitro with the peptide may be used to screen for peptide-specific helper T lymphocytes from a patient by measuring IFN-γ release. The present invention further relates to a method of preventing melanoma in a mammal, said method comprising administering to the mammal an effective amount of the TRP2 (180-188) peptide and or analogs thereof, alone or in combination with other HLA-A2-restricted epitopes from TRP2 or other melanoma antigens in an amount effective to prevent melanoma in the mammal. The present invention also relates to a method of preventing melanoma in a mammal, comprising administration of an effective amount of an HLA Class II restricted TRP2 peptide and/or analog thereof.

The present invention further relates to a method of preventing melanoma in a mammal, said method comprising administering to the mammal an effective amount of an expression vector containing nucleic acid sequences encoding the TRP2 (180-188) peptide and/or analogs thereof, alone or in combination with other HLA-A2-restricted epitopes from TRP2 or other melanoma antigens in an amount effective to prevent melanoma in the mammal.

The present invention also relates to a method of preventing melanoma in a mammal, said method comprising the administration of an effective amount of an expression vector containing nucleic acid sequences encoding an HLA-Class II restricted TRP2 peptide or analog thereof to prevent melanoma in the mammal.

In another embodiment, the nucleic acid sequence encoding at least one MHC Class II restricted peptide may be used directly as an immunogen or vaccine using techniques utilizing "naked" DNA which is directly injected into muscle or skin, or linked to a lipid molecule.

After immunization the efficacy of the vaccine can be assessed by production of immune cells that recognize the tumor antigen, as assessed by specific lytic activity, specific cytokine production, tumor regression or a combination of these approaches. If the mammal to be immunized is already afflicted with melanoma or metastatic melanoma, the vaccine can be administered in conjunction with other molecules such as immunomodulators, for example, IL-2, IL-6, IL-10, IL-12, IL-15, interferon, tumor necrosis factor and the like, adjuvants, chemotherapeutic drugs such as cisplatinum, antiviral such as gancyclovir, amphotericin B, antibiotics and the like. In the methods of preventing or treating melanoma, the HLA Class I restricted TRP2 (180-188) peptide and or analogs thereof or the HLA-Class II restricted TRP2 peptide and/or analog thereof may be administered via one of several routes including but not limited to subcutaneous, intradermal, intramuscular, intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, intratumor and the like. Administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be by nasal sprays, for example, or suppositories. For oral administration, the TRP2 peptide or analog thereof is formulated into conventional oral administration form such as capsules, tablets. A preferred route of administration of peptide is via subcutaneous injection in IF A (incomplete Freund's adjuvant). In general, it is desirable to provide the recipient with a dosage of TRP2 peptide or analogs thereof effective to prime, stimulate and/or cause the clonal expansion of peptide-specific T lymphocytes and/or helper T lymphocytes, preferably cytotoxic T lymphocytes, which in turn are capable of preventing or inhibiting melanoma in the recipient. A preferred dosage of TRP2 peptide or an analog thereof is of at least about 1 pg per kg bodyweight, more preferably at least about lng per kg bodyweight, and most preferably at least about lμg or greater per kg bodyweight of the recipient.

When nucleic acid of the invention is utilized in the method of preventing or treating melanoma, preferred routes of administration are intramuscularly and intradermally, and vectors containing the sequences are administered at a dosage of about 1 to about 10 mg.

The dose of peptide or nucleic acid is administered at least once and may be provided as a bolus or a continuous administration. Multiple administrations of the dose over a period of several weeks to months may be preferable. Subsequent doses may be administered as indicated. In general, it is desirable to provide DNA vectors at a dosage of about 1 to about 10 mg, at about 4 week intervals, and peptides at a dosage of about 1 mg, at about 3-4 week intervals.

The present invention also relates to a pharmaceutical composition comprising an HLA-Class I or HLA-Class II restricted TRP2 peptide and/or analogs thereof, alone or in combination with different HLA-A2-restricted epitopes from TRP2, or one or more different HLA-Class II restricted epitopes from TPR2, a combination thereof, or other melanoma antigens.

The present invention also relates to a pharmaceutical composition comprising an expressing vector comprising the nucleic acid sequence encoding an HLA-Class I or HLA-Class II restricted TRP2 peptide and/or analogs thereof, alone or in combination with HLA-A2-restricted epitopes from TRP2, an HLA-Class II restricted epitope from TRP2, or other melanoma antigens.

The TRP2 peptides of this invention or analogs thereof may be formulated alone or with any other HLA-A2-restricted epitopes from TRP2, an HLA- Class II restricted epitope from TRP2, or other melanoma antigens with pharmaceutically acceptable carriers into pharmaceutical compositions by methods known in the art. The compositions may further comprise at least one immunostimulatory molecule where immunostimulatory molecules to be used in conjunction with the TRP2 peptide for stimulating antigen specific T cell responses include, but are not limited to, one or more major histocompatibility complex (MHC) molecules, such as class I and class II molecules. The composition may further comprise other stimulator molecules including B7.1, B7.2, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72 and the like, and cytokines which include but are not limited to IL-1 through IL-15, TNFα, IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAP III, ENA-78, GRO, 1-309, PF-4, IP-10, LD-78, MGSA, MlP-lα, MIP-1 β, or combination thereof, and the like for immunopotentiation.

The present invention also encompasses antibody elicited by, and immunoreactive with, the HLA-Class II restricted TRP2 peptides. The antibody may be polyclonal, monoclonal, chimeric, or recombinant antibody. Recombinant antibody includes, but is not limited to single chain antibody which may be made by methods known in the art. The antibody of the present invention has utility as a diagnostic reagent in assays to detect cancer cells and in assays to monitor cancer vaccine therapy. The antibody may be provided in kit form along with other standard reagents for immunoassays. The antibody of the present invention may be used therapeutically in the form of a pharmaceutical composition to inhibit the growth of cancer cells expressing TRP2 peptides.

The present invention will now be described by way of examples, which are meant to illustrate, but not limit, the scope of the invention.

All of the patents and references cited herein are hereby incorporated by reference. Examples Materials and Methods Cell culture

The Skmel23 human melanoma cell line was kindly provided by Thierry Boon (Ludwig Institute for Cancer Research, Brussels, Belgium), and A375 was purchased from American Type Culture Collection (Rockville, MD). All other human melanoma cell lines were established in our laboratory (31). Melanoma cell lines and T2 cells (HLA-A*0201⁺ peptide transporter associated protein deficient T cell-B cell hybrid-(32) were routinely cultured in RPMI1640 (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Bio fluids, Rockville, MD) and 2 mM L-glutamine (Biofluids). The COS-7 monkey kidney cell line was kindly provided by W. Leonard (National Institutes of Health) and was maintained in DMEM (Mediatech or Biofluids) containing 10% heat-inactivated FBS, 2 mM L-glutamine, and 10 mM HEPES. Human lymphocytes were cultured in complete medium (CM) consisting of Iscove's modified DMEM with 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 mcg/ml streptomycin (Biofluids), and 10% heat-inactivated human AB serum (Sigma Chemical Co., St. Louis, MO; Valley Biomedical, Winchester, VA).

The presence of TRP2 mRNA in melanoma cell lines was previously assessed by Northern Blot analysis (13). The expression of HLA-A2 was evaluated by FACS using an anti-HLA-A2 monoclonal antibody (One Lambda, Canoga Park, CA), and for some cell lines, DNA sequencing confirmed the presence of HLA-A*0201 (HLA laboratory, National Institutes of Health, Bethesda, MD). The expression of TRP2, HLA-A2 or HLA-A*0201 in melanoma cell lines was as follows: 397mel (HLA-A2^", TRP2⁺), 888mel (HLA-A2^", TRP2⁺), A375 (HLA-A*0201⁺, TRP2 ),

526mel (HLA-A2⁺, TRP2⁺), 624mel (HLA-A*0201⁺, TRP2⁺), 1300mel (HLA-A2⁺, TRP2⁺), Skmel23 (HLA-A*0201⁺, TRP2⁺), and 501mel (HLA-A*0201⁺, TRP2⁺). In addition, COS-7 cells expressing HLA-A*0201 and TRP2 or MART-1 were generated by co-transfection (Lipofectamine Plus; Gibco) with cDNAs encoding these proteins (pCDNA3 plasmid; Invitrogen). Peptide synthesis and HLA-A *0201 binding affinity assays

Candidate peptides were selected from TRP2 which conformed to a permissive HLA-A*0201 binding motif were selected from TRP2 based on the following set of criteria: (1) peptides which bind with high affinity to HLA-A*0201 are generally 9 or 10 amino acids in length and contain L or M at the second position from the amino-terminus (P2) and V or L at the carboxy-terminus (P9 or P10) (30); (2) amino acids at secondary anchor positions, most notably PI and P3, can significantly affect peptide binding to HLA-A*0201 (28; 29); and (3) many previously identified HLA-A*0201 restricted epitopes from nonmutated melanoma antigens are 9 amino acids in length and have a dominant residue at P2 or the C-terminus, but not both primary anchor positions (37; 38). Based on these observations, 9-mers were chosen which contained L/M at P2 or V/L at P9; 10-mers were selected with L/M at P2 and L/V at P10; and peptides with D, E, P, R, or H at PI were eliminated. In total, 44 9- mers and 7 10-mers were synthesized (Table 1). TRP2 peptides and the FluMl peptide (influenza matrix protein (58-

66): GILGFNFTL: SEQ ID NO: 57) were initially synthesized using a solid-phase method based on fluorenylmethoxycarbonyl (Fmoc) chemistry on a multiple peptide synthesizer (model AMS 422; Gilson Co., Worthington, OH), and molecular weights were verified by laser desorption mass spectrometry (Bio-Synthesis, Inc., Lewisville, TX). MART-l(27-35) (AAGIGILTV: SEQ ID NO: 58) was commercially synthesized, purified (>95%), and characterized by amino acid analysis (Peptide Technologies, Gaithersburg, MD).

The relative binding affinity of each TRP2 peptide to HLA-A* 0201 was experimentally determined on the basis of the inhibition of binding of a standard radiolabeled peptide to purified MHC molecules as previously described (33). Briefly, the test peptide was coincubated at various concentrations (1 nM to 100 mM) with soluble HLA-A*0201 heavy chain, human β₂-microglobulin, and 5 nM ¹²⁵I-labeled HBc (18-27) with Y substituted at P6 (FLPSDYFPSN: SEQ ID NO: 59). The concentration of the test peptide necessary to inhibit the binding of the iodinated peptide by 50% (ID₅₀) was calculated. In later experiments, TRP2 (217-225) was purified (>95%) by reverse- phase HPLC on a POROS 10 column (PerSeptive Biosystems, Cambridge, MA) using a 0.05% trifluoroacetic acid/water-acetonitrile gradient, and the molecular weight was verified by mass spectrometry (Bio-Synthesis, Inc.). TRP2(180-188) and the FluMl peptide were commercially synthesized, purified (>95%), and characterized by amino acid analysis (Peptide Technologies).

The binding affinities of TRP2 peptides to HLA-A*0201 were evaluated on the basis of the inhibition of binding of a standard radiolabeled peptide to purified MHC molecules (33). The concentration of test peptide necessary to inhibit the binding of the standard peptide by 50% (ID₅₀) was calculated, and peptide binding affinity was defined as high (ID₅₀<50 nM), intermediate (50 nM<ID₅₀<500 nM), or weak (500 nM<ID₅₀).

Cytokine release and cytotoxicity assays

Recognition of TRP2 by bulk T cell cultures was evaluated about 7 days after each stimulation on the basis of IFNγ secretion by CTLs in response to T2 cells preincubated with peptide, HLA-A2⁺ TRP2⁺ melanoma cells, or, in some experiments, COS-7 cells expressing HLA-A*0201 and TRP2. T2 cells were incubated with peptide 1 to 3 hr at 37° C and were either used directly (peptide-loaded) or were washed twice prior to use (peptide-pulsed). 10⁵ responder T cells were coincubated with 10⁵ stimulator cells (250 μl total) about 20 hr at 37° C, and the concentration of human IFNγ in coculture supernatants was measured using a commercially available ELISA kit (Endogen, Cambridge, MA).

In some experiments, 4 hr ⁵¹Cr release cytotoxicity assays were also performed to evaluate the recognition of TRP2 by bulk CTL as previously described (36). Briefly, ⁵¹ Cr-labeled T2 cells were incubated with 1 μM peptide for about 1 hr at 37° C and washed twice. These cells and ⁵¹ Cr-labeled melanoma cells were coincubated with effector cells (peptide stimulated CTLs) (5000 targets/well; multiple E:T ratios; 150 μl total) 4 hr at 37° C, and the radioactivity in coculture supernatants was determined by gamma counting. The percent specific lysis of target cells by CTL was calculated, and spontaneous ⁵¹Cr release from target cells never exceeded 20% of the maximum.

Table 1 Binding Affinities of TRP2 Peptides to HLA-A*0201

Peptide Sequence ID No: Sequence IDso CnM)¹

431-439 5 NMVPFFPPV 4

185-193 6 FVWLHYYSV 15

455-463 7 YAIDLPVSV 16

288-296 8 SLDDYNHLV 26

482-490 9 ALVGLFVLL 26

476-484 10 VMGTLVALV 28

479-488 11 TLNALVGLFN 29

180-188 12 SNYDFFVWL 36

475-483 13 VVMGTLVAL 67

156-164 14 YVITTQHWL 104

217-225 15 VTWHRYHLL 161

360-368 16 TLDSQNMSL 161

367-376 17 SLHΝLNHSFL 263

472-481 18 TLLVVMGTLV 357

234-242 19 LIGΝESFAL 385

394-402 20 VLHSFTDAI 417

364-372 21 QVMSLHΝLV 676

216-224 22 FVTWHRYHL 943

473-481 23 LLVVMGTLV 1351

241-249 24 ALPYWNFAT 1351

489-498 25 LLAFLQYRRL 1515

472-480 26 TLLVVMGTL 2632

450459 27 QLGYSYAIDL 2632

9-17 28 LLSCLGCKI 3571

385-393 29 SAANDPIFV 4545

478-486 30 GTLVALVGL 4545

28-36 31 VCMTVDSLV 7143

481-489 32 VALVGLFVL 7143

406-414 33 WMKRFNPPA 8333

20-28 34 GAQGQFPRV >10,000

56-64 35 QGRGQCTEV >10,000

117-125 36 NCERKKPPV >10,000

125-133 37 VIRQNIHSL >10,000

144-152 38 ALDLAKKRV >10,000

158-166 39 ITTQHWLGL >10,000

159-167 40 TTQHWLGLL >10,000

Peptide binding affnity to HLA-A*0201 was evaluated by measuring the concentration of peptide necessary to inhibit the binding of a standard radiolabeled peptide by 50% (IDso). Relative binding affnity was defined as high (I DsO<50 nM), intermediate (50 n M< I DsO<500 nM ), or weak (ID50>500 nM). Peptide Sequence ID No: Sequence ID₅₀ (nM)¹

163-171 41 WLGLLGPNG >10,000

178-186 42 NCSVYDFFV >10,000

226-234 43 CLERDLQRL >10,000

248-256 44 ATGRNECDV >10,000

264-272 45 AARPDDPTL >10,000

311-319 46 QMGRNSMKL > 10,000

321-329 47 TLKDIRDCL >10,000

343-351 48 STFSFRNAL >10,000

386-394 49 AANDPIFVV >10,000

480-488 50 LVALVGLFV >10,000

485-493 51 GLFVLLAFL >10,000

490-498 52 LAFLQYRRL >10,000

502-510 53 YTPLMETHL >10,000

9-18 54 LLSCLGCKIL >10,000

76-87 55 ILRNQDDREL >10,000

Example 1 Identification of HLA-A*0201 Binding Peptides From TRP2 Of the 51 TRP2 peptides which fit the extended HLA-A* 0201 binding motif as described in the Methods section, only 16 actually bound to HLA-A*0201 with high (ID₅₀<50 nM)or intermediate affinity (50 nM<ID₅₀<500 nM) (Table 1).

Example 2 CTL Induction with TRP2 Synthetic Peptides In Vitro The 21 TRP2 peptides with ID₅₀<2000 nM (Table 1) were used to stimulate peripheral blood lymphocytes (PBL) in vitro from 4 HLA-A*0201⁺ patients with metastatic melanoma.

In an initial screening of peptides for the induction of tumor reactive CTL, the 21 TRP2 peptides and MART-l(27-35) (as a positive control) were used to stimulate lymphocytes in vitro from 4 HLA-A*0201⁺ melanoma patients as follows: T cell cultures were established by plating peripheral blood mononuclear cells (PBMC) in 24 well plates (1.5xl0⁶ cells/ml; 2 ml/well) in CM containing peptide (5 μg/ml for TRP2 peptides or 2 μg/ml for MART- 1(27-35)) and GM-CSF (200 U/ml; PeproTech, Rocky Hill, NJ) and IL-4 (100 U/ml; PeproTech) to promote the differentiation of dendritic cells (34). Seven days later, 300 IU/ml of rIL-2 (Chiron Co., Emeryville, CA) was added. On day 11 and weekly thereafter, lymphocytes were restimulated with peptide-pulsed autologous PBMC as previously described (35).² LFNγ secretion in response to peptide-loaded T2 cells and HLA-A2⁺ TRP2⁺ melanomas was measured 7 days after the third and fourth restimulations (days 32 and 39).

Lymphocytes from 3 of 4 patients proliferated during this study, and for a given patient, no significant or consistent differences were observed between lymphocyte expansions with different peptides. Peptide recognition by bulk T cell

2

Briefly, responder lymphocytes were harvested and replated in new 24-well plates (2 5xl0⁵ cells/ml, 2 ml/well) m CM

Autologous irradiated (3000 rad) PBMC were incubated with peptide (₅ μg/ml for TRP2 peptides or 2 μg/ml for MART-1(27- 3₅)) in 1 ml conical tubes (l-3xl0⁶ cells/ml, 6-12 ml/tube) 2 to 4 hours at 37° C Peptide-loaded PBMC were washed and added to responder lymphocytes at a responder to stimulator ratio of about 1 7 One day after each restimulation, 150 IU/ml rIL-2 was added, and generally, cultures were split 1 1 two to three days later with CM containing 300 IU/ml rIL-2 cultures after 4 restimulations is presented in Table 2. In patients HU and IN, TRP2 (431-439), TRP2 (180-188), TRP2 (217-225), and MART-1 (27-35) induced peptide reactive T cells, and in patient CA, peptide specific CTL were generated with TRP2 (476-484) and TRP2 (217-225). However, no TRP2 peptide induced CTL specifically recognized HLA-A2⁺ TRP2⁺ melanoma cells; only T cells from patients HU and IN stimulated with the positive control peptide MART-1 (27-35) specifically secreted IFNγ in response to HLA-A2⁺ melanomas (data not shown).

One possible explanation for the lack of melanoma recognition by bulk T cells stimulated with TRP2 peptides was that these cultures contained only small numbers of high avidity T cells capable of recognizing low levels of TRP2 peptides expressed on the surfaces of melanoma cells. If this were the case, melanoma reactivity might be apparent in T cell clones derived from peptide specific cultures.

However, since these T cells were unavailable for further analysis, PBL from additional HLA-A*0201⁺ melanoma patients were stimulated in vitro with the two TRP2 peptides which most efficiently induced peptide-reactive CTL in the initial screening: PBL from 4 HLA-A*0201⁺ melanoma patients (MU, KU, AN, and WE) were stimulated in vitro with TRP2(180-188), and PBL from 4 separate patients (MC, WO, LE, and CA) were stimulated with TRP2(217-225) using a standard protocol which had previously been used successfully to generate melanoma reactive CTL with peptides from MART-1 and gplOO (40, 41).

In particular, PBMC were initially cultured in culture medium containing 1 μM peptide (instead of 5 μg/ml) without GM-CSF and IL-4, and 300 IU/ml IL-2 was added two days later. The lymphocytes were then restimulated weekly with peptide-pulsed autologous PBMC as described above (see footnote 2) beginning at day 7 (instead of day 1 las in the initial screening), except that the PBMC were added to the responsive lymphocytes at a responder to stimulator ratio of about 1:10. IFNγ secretion in response to peptide-loaded T2 cells, COS-7 cells expressing HLA- A*0201 and TRP2, and HLA-A2⁺ TRP2⁺ melanoma cells was measured about 7 days after each stimulation beginning at one week. U

*IFNγ release (pg/ml) in 20 hr coculture supernatants of stimulators with bulk T cells after 4 in vitro restimulations with the indicated peptide.

Jmedia: LFNγ release in the absence of stimulators.

§FluMl: LFNγ release in response to T2 cells preincubated with 5 g/ml FluMl peptide.

Tfrelevant peptide: IFNγ release in response to T2 cells preincubated with 5 g/ml of the peptide used for PBL sensitization.

**underlined values indicate that LFNγ release in response to T2 cells preincubated with the relevant peptide was >50 pg/ml and at least twice background with either media or T2 cells pre-loaded with the FluMl peptide.

Specific peptide recognition by bulk CTL stimulated with TRP2(217- 225) was apparent as early as week 3 from one patient (WO), and by week 5, T cell cultures from 3 of the 4 patients (MC, WO, and LE) specifically released IFNγ in response to peptide-loaded T2 cells. Similar to the initial TRP2 peptide screening, none of the bulk cultures stimulated with TRP2(217-225) recognized COS-7 transfectants or HLA-A2⁺ TRP2⁺ melanoma cells (data not shown).

TRP2 (180-188) induced peptide reactive T cells from 2 of 4 patients (KU and MU) after only 2 restimulations (week 3). More significantly, at week 3, bulk CTL from patient KU specifically secreted IFNγ when cocultured with COS-7 cells expressing HLA-A*0201 and TRP2 and HLA-A2⁺ TRP2⁺ melanoma cells. By week 5, the same pattern of recognition was observed by CTL from patient MU. Based on these observations, lymphocytes from the 3 patients whose cells proliferated (AN, KU, and MU) were restimulated, and by week 7, bulk CTL from all three patients specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and TRP2, and HLA-A2⁺ TRP2⁺ melanomas (Table 3).

To determine if recognition of melanoma cells correlated with that of low concentrations of peptide, IFNγ release by CTL was measured in response to T2 cells pulsed with various concentrations of TRP2 (180-188) between 10^'5 and 10^"12 M. Peptide-induced CTL from patients KU and MU recognized TRP2 (180-188) pulsed on T2 cells at a concentration of 10^"9 M, whereas T cells from patient AN did not specifically release IFNγ at a peptide concentration below 10^"7 M. This correlated with specific tumor recognition in that IFNγ release by T cells from patient AN in response to HLA-A2⁺ TRP2⁺ melanoma cells was generally lower than that from patients KU and MU (Table 3). Table 3

Recognition of Peptide, CoS-7 Transfectants. and Melanoma Cells by PBL from HLA-A*0201⁺ Melanoma Patients Stimulated with TRP2(180-188)^*

Patient: MU Patient: KU Patient: AN

Media 0 12 o ^•

T2 + nopeptide 0 8 0

T2 + 1 μM FluMl 0 nt^J 0

T2 + 10^'I2 M TRP2(180-188) 0 nt 0

T2 + 10-ⁿ M TRP2(180-188) 2 nt 0

T2 + 10^"10 M TRP2 (180-188) 18 nt 11

T2 + 10^'9 M TRP2 (180-188) 597^§ 130 23

T2 + 10-⁸ M TRP2 (180-188) 1112 455 38

T2 + 10^'7M TRP2 (180-188) 3476 3298 385

T2 + 10^"6 M TRP2 (180-188) 4107 5973 584

T2 + 10^"5 M TRP2 (180-188) 4132 7295 622

COS-A2-MART 0 11 0

397mel (Al, TRP2⁺) 0 13 0

888mel (A2^", TRP2⁺) 0 14 0

A375mel (A2⁺, TRP2 ) 1 15 8

501 mel (A2\ TRP2⁺) 1946 nt 230

526mel (A2⁺, TRP2⁺) 1890 858 207

624mel (A2⁺, TRP2⁺) 1839 451 139

1300mel (A2⁺, TRP2⁺) 1646 147 51

Sk23mel (A2⁺, TRP⁺) 1336 50 17

^* IFNγ release (pg/ml) in 20 hr coculture supernatants of target cells with bulk T cell cultures after 6 in vitro restimulations with TRP2 (180-188). ⁱ nt indicates not tested due to low numbers of CTL

^§ underlined values indicate that IFNγ release in response to T2 cells preincubated with TRP2(180-188) or HLA-A2⁺ TRP2⁺ targets was >50 pg/ml and at least twice background with any HLA-A2^" or TRP2^" target. Specific lysis of peptide-pulsed T2 cells and melanomas by CTL induced with TRP2 (180-188) was also measured at week 7 in 4-hr ⁵¹Cr-release cytotoxicity assays. CTL from all 3 patients specifically lysed T2 cell pulsed with 1 μM TRP2 (180-188) compared to T2 cells without exogenous peptide (Fig. 1 a-c). Furthermore, in comparison to 2 HLA-A2^" TRP2⁺ and one HLA-A*0201⁺ TRP2^" melanoma lines, all T cell cultures specifically lysed 3 HLA-A2⁺ TRP2⁺ melanomas (Fig. 1 d-f).

Discussion The lack of specific melanoma recognition by bulk T cell cultures stimulated with TRP2(180-188) in the initial peptide screening in Example 2 may have been due to a technical difference between that experiment and the latter. In particular, in the later screening in Example 2, PBL were stimulated with peptides in vitro using a protocol which had been used successfully to generate melanoma reactive CTL with MART-1 and gplOO peptides (40, 41). This protocol differed from that used in the initial peptide screening in that GM-CSF and IL-4 were absent in the initial culture period, and the first restimulation was performed on day 7 as opposed to day 11. In addition, in the second set of CTL inductions, a lower concentration of peptide was used (1 μM vs. 5 μg/ml). This may account for the induction of melanoma reactive CTL in the latter experiment but not the former since in a previous study, high-avidity CTL capable of clearing a viral infection in mice could only be generated in vitro using comparatively low concentrations of peptide (47). Another potential explanation was that a lower responder to stimulator ratio was generally used for restimulations in the preliminary TRP2 peptide screening than in the later CTL inductions (average 1:7 vs. 1:10) due to the large numbers of autologous PBMC needed to restimulate cultures with 22 different peptides. In Example 2, specific recognition of TRP2(180-188) preceded that of tumor by 1-3 weeks. Therefore, tumor reactivity may have become apparent in the initial screening with TRP2(180-188) if additional restimulations had been performed. Conclusions The above Examples describe the identification of TRP2 (180-188) (SVYDFFVWL) as a new HLA-A*0201 restricted T cell epitope capable of inducing melanoma reactive CTL. TRP2 is a melanosomal enzyme expressed in most mammalian melanocytic cells and may represent an ideal target antigen for the immunotherapeutic treatment of patients with melanoma. This protein has previously been identified as a melanoma antigen recognized by tumor reactive T cells in the context of HLA-A31 (13) and HLA-A33 (14). However, the frequencies of these alleles among melanoma patients in the United States are low compared to that of HLA-A*0201 , which is the most commonly expressed class I HLA allele in the

Caucasian population of North America (about 6% for HLA- A31 and about 2% for HLA-A33 compared to about 46% for HLA-A*0201) (15). Therefore, the identification of TRP2(180-188) as a new HLA-A*0201 restricted T cell epitope will enable the treatment of a much larger group of melanoma patients with TRP2 based immuno therapies.

Furthermore, TRP2(180-188) may be valuable for the development of vaccines for the treatment of patients diverse in HLA expression since a supermotif has been defined for a family of MHC molecules including 4 subtypes of HLA-A2 (HLA- A*0201, HLA-A*0202, HLA-A*0205, and HLA-A*0206) and two independent class I HLA molecules (HLA-A*6802 and HLA-A*6901 ) which bind small peptides with aliphatic residues at P2 and the C-terminus (67), and TRP2(180-188) conforms to this supermotif since it contains L at P9 and V at P2. TRP2(180-188) may therefore bind with high affinity to 6 class I MHC molecules in addition to HLA-A*0201 and H-2K^b (unpublished data). In addition, using a statistical algorithm to predict the half time of dissociation of a peptide-MHC-β2 microglobulin complex (68), of all 511 possible 9- mers from TRP2, the TRP2(180-188) peptide was predicted to be within the top 25 highest binding affinity peptides for HLA-A*0201 (ranked #2), HLA-A*0205 (ranked #1), HLA-A3 (#22), HLA-B7 (#14), HLA-B*3901 (#8), HLA-Cw*0301 (#1), and HLA-Cw*0602 (#12). These predictions therefore provide further evidence that TRP2(180-188) is likely to be presented on the surfaces of melanomas expressing a wide variety of class I HLA molecules.

Example 3 Identification of an antigen recognized by melanoma reactive CD4⁺ T cells

Initial studies demonstrated that a CD4⁺ T cell clone 7, isolated from patient 1290 diagnosed with melanoma recognized tumor cell lysates incubated with autologous Epstein Barr Virus (EBV) B cells (888 EBV B). In addition, this clone recognized a number of allogeneic melanomas that shared expression of the HLA- DRB1*1501 or 1502 alleles (Table 4).

Table 4

Stimulator IFN-γ (pa/mi)

888 EBV B 47

697 mel (IFN-γ)¹'² 25,100

1011 mel (LFN-γ)¹'² 44,100

1290 mel (IFN-γ)¹'² 3200

1290 fibroblast 55

None 97

¹ Tumor cells were treated for 48 hours with interferon gamma (LFN-γ) before use as stimulators.

² 1290 mel cell express DRB1*1502, and 697 and 1011 mel express DRB1*1501.

The monkey kidney cell line COS was transfected with cDNAs that encoded a number of the melanocyte antigens that had previously been shown to be recognized by HLA class I restricted T cells. When the autologous EBV B cells were pulsed with COS transfectants, it was found that cells that had been transfected with a construct encoding human TRP-2 were stimulatory (Table 5). In addition, purified recombinant TRP-2 protein was recognized by clone 7 T cells (Table 6). Table 5

Stimulator Transfected lysate IFN -γ(pg ml)

20 ul lvsate lO ul lvsate

888 EBV B - 31 37 pCDNA-GFP 18 14 pCDNA-β-catenin 14 16 pCDNA-TRP-1 19 24 pCDNA-TRP-2 201 151 pCDNA-501 tyr. 15 19 pCDNA-MART-1 26 20 pCDNA-gplOO 38 18

888 mel 99 98

1290 mel (IFN-γ)¹ >5000

None 23

¹ Tumor cells were treated for 48 hours with interferon gamma (IFN-γ) before use as stimulators.

Table 6

Protein μg/ml IFN-γ roε/mn

TPR-2¹ 1 180

0.5 139

0.2 47

0.1 24

0.05 14 pglOO 100 <8

50 <8

25 <8

10 <8

5 <8

None _ <8

¹ Proteins were pulsed overnight at the indicated concentrations on 888 EBV B cells, and the following day 2 X 10⁴ T cells were incubated with the pulsed and unpulsed B cells.

A series of overlapping 20-mer peptides from the human TRP-2 molecule that overlapped at 10 amino acids were then tested for recognition by clone 7 T cells following incubation with autologous EBV B cells. Two peptides (LQRLIGNESFALPYWNFATG and ALPYWNFATGRNECDVCTDQ) containing the overlapping sequence ALPYWNFATG stimulated 330 and 444 pg/ml of IFY-γ from clone 7 T cells, whereas all of the other TRP-2 peptides stimulated less than 15 pg/ml of LFY-γ. Additional studies aimed at determining the minimal epitope as well as optimizing the epitope based on the binding motif for HLA-DRB 1*1501 have also been carried out (Table 7).

Table 7

Peptide Sequence IFN-γ (pg/ml) Neat 1:10

A5 ALPYWNFATGRNECDVCTDQ >500 3250

F4 LQRLIGNESFALPYWNFATG >500 880

A5 VLPYWNFATG >500 890

B4 FALPYWNFATG >500 1600

C4 ALPYWNFATGR >500 820

D4 QLPYWNFATG >500 4600

D7 LPYWNFATG >500 570

E4 SLPYWNFATG >500 1700

E7 ALPYWNFAT 337 1100

F4 ALPYWNFATG >500 1100

None _ 46

In this preliminary assay, it appears that the minimal epitope required for recognition by clone 7 T cells is ALPYWNFAT. The attempt to modify this peptide according to the published motif (Rammensee et al Immunogenetics 1995, 41 :178-228) does not appear to have generated any peptides that are significantly better than the parental peptide. This motif consists of a L, V or I residue at position 1 , a F, Y or I residue at position 4, and then an I, L, V, M or F residue at position 7. In one embodiment, analogs were generated assuming that the first A residue represents position 1 in the peptide. In another embodiment, the L residue represents position 1 and analog peptides containing a modification of the W at position 5 in this peptide and the A at position 8 have been synthesized and are being tested for recognition by clone 7 T cells.

In addition, a peptide tested in this assay, SLPYWNFATG is derived from the sequence of TRP-1, a gene product with a very similar sequence to TRP-2. The substitution of S for A at the first position in the sequence does not appear to affect recognition, implying that recognition of cells expressing these gene products would be very similar. This in fact was shown to be true by the transfection of autologous EBV B cell with constructs encoding TRP-1 and TRP-2 (Table 8). Recognition of both gene products was observed using fusion constructs containing the invariant chain (Ii) amino terminal 80 amino acids, which was used to target these products to the class II presentation pathway. In addition, recognition of the TRP-1 and TRP-2 gene products was also observed in the absence of the Ii targeting sequence.

Table 8

Stimulators

888 EBV B transfected with: LFN-v Cpe/mD

PEAK-GFP 26

PEAK-Ii-gplOO 30

PEAK-Ii-tyrosinase 18

PEAK-Ii-TRP-1 >1000

PEAK-TRP-1 120

PEAK-Ii-TRP-2 800

PEAK-TRP-2 700

1290 mel >500

1290 mel-IFN-γ >500 References

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Claims

1. An isolated peptide having an amino acid sequence according to SEQ ID NO:l or analogs thereof.

2. The peptide of claim 1, wherein said peptide has an amino acid sequence according to SEQ ID NO: 1.

3. The peptide of claim 1, wherein said peptide is an analog of SEQ ID NO: 1 in which valine at position 2 of SEQ ID NO: 1 is substituted with either leucine or methionine.

4. A nucleic acid molecule encoding the peptide of claim 1.

5. The nucleic acid molecule of claim 4, wherein the molecule has a sequence according to SEQ ID NO:2.

6. An expression vector comprising the nucleic acid molecule of claim 4.

7. A host cell comprising the vector according to claim 6.

8. The peptide according to claim 1 , wherein the peptide is immunologically recognized by HLA-A2 restricted T lymphocytes.

9. The peptide according to claim 8, wherein the peptide is immunologically recognized by HLA-A*0201 restricted T lymphocytes.

10. A method of detecting the presence of melanoma in a mammal comprising:

(a) coincubating T lymphocytes isolated from the mammal with TRP2⁺ target cells or with target cells pre-exposed to the peptide of claim 1 ;

(b) measuring the response of the T lymphocytes to the target cells in step (a); and (c) comparing the response of the T lymphocytes as measured in step (b) with the response of said mammalian T lymphocytes coincubated with control target cells, wherein a two-fold or more increase in response of the T lymphocytes as measured in step (b) as compared to the response of the T lymphocytes coincubated with control target cells indicates the presence of melanoma in said mammal

11. The method of claim 10, wherein the target cells are selected from the group consisting of HLA-A2⁺TRP2⁺ melanoma cells, HLA-A*0201 T2 cells, dendritic cells and PBMCs.

12. The method of claim 10, wherein the response of the T lymphocytes is measured by determining cytolytic activity of the T lymphocytes towards the target cells.

13. The method of claim 12, wherein the cytolytic activity is measured by a ⁵¹Cr release assay.

14. The method of claim 10, wherein the response of the T lymphocytes is measured by assaying cytokine release by the T lymphocytes.

15. The method of claim 13 , wherein the cytokine release by the T lymphocytes is determined by measuring IFNγ secretion by the T lymphocytes.

16. A method of treating a mammal having melanoma, said method comprising: (a) exposing T lymphocytes in vitro to a peptide of claim 1, alone, or in combination with other HLA- A2 -restricted melanoma antigen epitopes to elicit peptide-specific cytotoxic T lymphocytes; and (b) administering the peptide-specific cytotoxic T lymphocytes of (a) to the mammal in a therapeutically effective amount.

17. The method of claim 16, wherein the other HLA-A2 restricted epitopes are selected from the group consisting of epitopes from MART-1, gplOO, tyrosinase, MAGE-3, GnT-V, and MC1R melanoma antigens.

18. A method of treating a mammal with melanoma comprising administering to said mammal a therapeutically effective amount the peptide of claim 1 , alone or in combination with expression vector encoding other HLA-A2 restricted melanoma antigen epitopes.

19. A method of treating a mammal with melanoma comprising administering to said mammal a therapeutically effective amount of the vector of claim 6, alone or in combination with expression vectors encoding other HLA-A2 restricted melanoma antigen epitopes.

20. A method of treating a mammal with melanoma comprising administering to the mammal a therapeutically effective amount of target cells pre- exposed to the peptide of claim 1.

21. A method of preventing melanoma in a mammal comprising administering to the mammal the peptide of claim 1 , alone or in combination with other HLA-A2-restricted melanoma antigen epitopes, in an amount effective to prevent melanoma in a mammal.

22. The method of claims 18, 19, 20 or 21, wherein the other HLA- A2 restricted epitopes are selected from the group consisting of MART-1, gplOO, tyrosinase, MAGE-3, GnT-N, and MC1R melanoma antigens.

23. A pharmaceutical composition comprising the peptide of claim 1 , alone or in combination with other HLA- A2 -restricted melanoma antigen epitopes, and a suitable excipient, diluent or carrier.

24. The pharmaceutical composition of claim 23, wherein the other HLA-A2 restricted epitopes are selected from the group consisting of MART-1, gplOO, tyrosinase, MAGE-3, GnT-V, and MC1R melanoma antigens.

25. Target cells exposed in vitro to the peptide of claim 1.

26. A pharmaceutical composition comprising the target cells of claim 25.

27. A method for obtaining T lymphocytes which exhibit a specific response to an HLA-Class I restricted TRP2 peptide, said method comprising administering target cells of claim 25 to a mammal in an amount effective to elicit a T lymphocyte response to the peptide.

28. A method for obtaining T lymphocytes which exhibit a specific response to an HLA-Class I restricted TRP2 peptide, said method comprising administering the peptide of claim 1 or a vector expressing the peptide of claim 1 to a mammal in an amount effective to elicit a peptide-specific T lymphocyte response.

29. T lymphocytes prepared by the method of claim 27.

30. T lymphocytes prepared by the method of claim 28.

31. A pharmaceutical composition comprising the T lymphocytes of claim 29.

32. A pharmaceutical composition comprising the T lymphocytes of claim 30.

33. An isolated peptide comprising an amino acid sequence

Xaa, LPYWNFATXaa-, of SEQ ID NO: 60 or analogs thereof, wherein Xaa, is any one of 20 naturally occurring amino acids or no amino acid and Xaa₂ is any one of 20 naturally occurring amino acids or no amino acid.

34. The peptide of claim 33, wherein said peptide is extended by about one to about 11 amino acids at the N-terminus, a C-terminus or combination thereof.

35. The peptide of claim 33, wherein the peptide comprises the amino acid sequence Xaa₃Xaa₁LPYWNFATXaa₂Xaa₄ (SEQ ID NO: 71), wherein Xaa, is any one of 20 naturally occurring amino acids or no amino acid, and Xaaj is any one of 20 naturally occurring amino acids or no amino acid; Xaa₃ comprises variably from zero to about 11 amino acids in length; and Xaa₄ comprises variably from zero to about 11 amino acids in length.

36. The isolated peptide of claim 33, wherein the amino acid sequence comprises: LPYWNFATG (SEQ ID NO: 61); ALPYWNFAT (SEQ ID NO:62); ALPYWNFATG (SEQ ID NO: 63); SLPYWNFATG (SEQ ID NO: 64);

QLPYWNFATG (SEQ ID NO: 65); VLPYWNFATG (SEQ ID NO: 66);

ALPYWNFATGR (SEQ ID NO: 67); FALPYWNFATG (SEQ ID NO: 68);

LQRLIGNESFALPYWNFATG (SEQ ID NO: 69); ALPYWNFATGRNECDVCTDQ

(SEQ ID NO: 70) or analogs of each sequence.

37. The peptide of claim 33, wherein Tyr is replaced by Phe or He.

38. The peptide of claim 33, wherein Phe is replaced by He, Leu, Val or Met.

39. The peptide of claim 33, wherein Tyr is replaced by Phe or He and Phe is replaced by He, Leu, Val or Met.

40. The peptide of claim 33, wherein Tip is replaced by Phe, Tyr or He.

41. The peptide of claim 33, wherein Ala is replaced by He, Leu, Val, Met or Phe.

42. The peptide of claim 33, wherein Tip is replaced by Phe, Tyr or

He and Ala is replaced by He, Leu, Val, Met or Phe.

43. The peptide according to any of claims 33 through 42, wherein the peptide is immunologically recognized by HLA-DR restricted T lymphocytes.

44. The peptide according to claim 43, wherein the peptide is immunologically recognized by HLA-DRB 1 *-l 501 restricted T lymphocytes or by HLA-DRB* 1502 restricted T lymphocytes.

45. A pharmaceutical composition comprising at least one peptide of any of claims 33 to 44 and a suitable excipient, diluent or carrier.

46. The pharmaceutical composition according to claim 45 in combination with an HLA-DR molecule.

47. A nucleic acid molecule encoding the peptide of any of claims 33 through 44.

48. An expression vector comprising the nucleic acid molecule of claim 47.

49. The vector according to claim 48, wherein the vector is a recombinant virus.

50. A host cell transformed or transfected with the vector according to claim 49.

51. The host cell according to claim 50 further comprising an HLA- DR molecule.

52. A method of detecting the presence of melanoma in a mammal comprising:

(a) coincubating T lymphocytes isolated from the mammal with target cells pre-exposed to the peptide of claim 33; (b) measuring the response of the T lymphocytes to the target cells in step (a); and

(c) comparing the response of the T lymphocytes as measured in step (b) with the response of said mammalian T lymphocytes coincubated with control target cells, wherein a two-fold or more increase in response of the T lymphocytes as measured in step (b) as compared to the response of the T lymphocytes coincubated with control target cells indicates the presence of melanoma in said mammal.

53. The method of claim 52, wherein the target cells are selected from from HLA-DR⁺B cells, dendritic cells or PBMCs.

54. The method of claim 52, wherein the response of the T lymphocytes is measured by assaying cytokine release by the T lymphocytes.

55. The method of claim 54, wherein the cytokine release by the T lymphocytes is determined by measuring IFNγ secretion by the T lymphocytes.

56. A method of treating a mammal having melanoma, said method comprising:

(a) exposing T lymphocytes in vitro to a peptide of claim 33, alone, or in combination with other HLA-DR-restricted melanoma antigen epitopes to elicit peptide-specific helper T lymphocytes; and

(b) administering the peptide-specific helper T lymphocytes of (a) to the mammal in a therapeutically effective amount.

57. A method of treating a mammal with melanoma comprising administering to said mammal a therapeutically effective amount of the peptide of claim 1 , alone or in combination with another HLA-Class II restricted melanoma antigen epitope.

58. A method of treating a mammal with melanoma comprising administering to said mammal a therapeutically effective amount of the vector of claim 48, alone or in combination with expression vectors encoding other HLA-Class II restricted melanoma antigen epitopes.

59. A method of treating a mammal with melanoma comprising administering to the mammal a therapeutically effective amount of target cells pre- exposed to the peptide of claim 33.

60. A method of preventing melanoma in a mammal comprising administering to the mammal the peptide of claim 33, alone or in combination with other HLA-Class II restricted melanoma antigen epitopes, in an amount effective to prevent melanoma in a mammal.

61. Target cells exposed in vitro to the peptide of any of claims 33 through 44.

62. The target cells according to claim 61 , wherein the target cells express an endogenous or recombinant HLA-DRB 1* 1501 or HLA-DRB1*1502 molecule.

63. The target cells according to any of claims 61 through 62 wherein the target cells are selected from the group consisting of melanoma cells or antigen presenting cells.

64. A pharmaceutical composition comprising the target cells of any of claims 61 to 63.

65. A method for obtaining T lymphocytes which exhibit a specific response to an HLA-Class II restricted TRP2 peptide, said method comprising administering target cells according to any of claims 61-63 to a mammal in an amount effective to elicit a specific T lymphocyte response.

66. A method for obtaining T lymphocytes which exhibit a specific response to an HLA-Class II restricted TRP2 peptide, said method comprising administering the peptide of claim 33 or a vector encoding the peptide of claim 33 to a mammal in an amount effective to elicit a specific T lymphocyte response.

67. T lymphocytes prepared by the method of claim 65.

68. T lymphocytes prepared by the method of claim 66.

69. A pharmaceutical composition comprising the T lymphocytes of claim 67.

70. A pharmaceutical composition comprising the T lymphocytes of claim 68.

71. A antibody elicited by and immunoreactive with the peptide according to claim 33.

72. A method of monitoring the efficacy of a cancer vaccine therapy in a mammal comprising (A) isolating lymphocytes from the vaccine-treated mammal, (B) measuring immunoreactivity of the T lymphocytes in the presence of a peptide according to any of claims 33 through 44, an enhancement of immunoreactivity in comparison to immunoreactivity of control lymphocytes is indicative of efficacy.

73. Use of a peptide according to any of claims 1-3 or 33 to 44 for use in a method of treating a mammal having melanoma.

74. Use of a nucleic acid molecule according to any of claims 4, 5 or 47 for use in a method of treating a mammal having melanoma.

75. An immunogen comprising a peptide according to any of claims 1-3 or 33 to 44.

76. A cancer vaccine comprising a peptide according to any of claims 1-3 or 33 to 44, or combination thereof, alone or in combination with a cytokine or adjuvant.

77. A kit for diagnosing melanoma comprising at least one peptide according to any of claims 1-3, 33 to 44, or combinations thereof.