CLASS I MHC PHOSPHOPEPTIDES FOR CANCER IMMUNOTHERAPY AND DIAGNOSIS

Abstract

A set of phosphorylated peptides are presented by HLA A*0101, A*0201, A*0301, B*4402, B*2705, B*1402, and el B*0702 on the surface of melanoma cells. They have the potential to (a) stimulate an immune response to the cancer, (b) to function as immunotherapeutics in adoptive T-cell therapy or as a vaccine, (c) to facilitate antibody recognition of the tumor boundaries in surgical pathology samples, and (d) act as biomarkers for early detection of the disease. Phosphorylated peptides are also presented for other cancers.

Claims

1. A composition comprising one or more recombinant or synthetic peptides, wherein each recombinant or synthetic peptide is 8 to 50 amino acid residues long, and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-1391.

2.-7. (canceled)

8. The composition of claim 1, further comprising an adjuvant, a pH stabilizing agent, a wetting agent or an emulsifying agent.

9.-26. (canceled)

27. An antibody that specifically binds to a recombinant or synthetic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 1-1391.

28.-50. (canceled)

51. The composition of claim 1, which comprises an adjuvant.

52. The composition of claim 51, wherein the adjuvant is selected from the group consisting of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, dinitrophenol, bacille Calmette-Guerin (BCG), and Corynebacterium parvum, or combinations thereof.

53. The composition of claim 51, wherein the composition stimulates a T cell mediated immune response to at least one of the synthetic peptides when administered to a subject.

54. The composition of claim 1, wherein at least one of the recombinant or synthetic peptides is a phosphopeptide or a phosphopeptide mimetic.

55. The composition of claim 54, wherein the phosphopeptide mimetic comprises a mimetic of phosphoserine, phosphothreonine, or phosphotyrosine.

56. The composition of claim 55, wherein the mimetic of phosphoserine, phosphothreonine, or phosphotyrosine comprises a phosphorous atom is linked to the serine, threonine, or tyrosine amino acid residue through a carbon atom.

57. The composition of claim 56, wherein the mimetic of phosphoserine, phosphothreonine, or phosphotyrosine comprises a —CF.sub.2—PO.sub.3H group.

58. The composition of claim 56, wherein the mimetic of phosphoserine, phosphothreonine, or phosphotyrosine comprises a —CH.sub.2—PO.sub.3H group.

59. An in vitro composition comprising dendritic cells loaded with one or more recombinant or synthetic peptides, wherein each recombinant or synthetic peptide: (i) is 8 to 50 amino acids long; and (ii) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-1391.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A and 1B are a graphic illustration of the recognition of naturally processed and presented phosphorylated peptides on cancer cells by the phosphopeptide-specific CTL. Phosphopeptide-specific CTL were incubated with the following cancer cell lines or EBV-transformed B lymphoblastoid cell lines (BLCL): COV413.AAD.A4 ovarian carcinoma, DM331.AAD.A4 and SLM2.AAD.A1 melanomas, MCF7.AAD.A2 and MDAMB231.AAD breast carcinomas, and JY EBV-BLCL. Supernatants were harvested and evaluated for the presence of murine IFNγ (produced by murine CTL lines). As a positive control, cancer cells were pulsed with the specific phosphopeptide to show that they are capable of presenting exogenously added peptide. In FIG. 1A, two phosphopeptide-specific CTL cell lines, 6850 and 6960 that are specific for the phosphopeptide GLLGpSPVRA (SEQ ID NO: 1268), recognize the phosphopeptide on all the cancer cell lines, but not the control cell line. In FIG. 1B, two phosphopeptide-specific CTL cell lines, 5183 and 63 that are specific for the phosphopeptide RVApSPTSGV (SEQ ID NO: 1289), recognize the phosphopeptide on all the cancer cell lines, but not the control cell line. The designation “pS” denotes a phosphoserine residue. The ordinate indicates murine IFNγ in pg/ml. The abscissa indicates each cell line.

[0022] FIGS. 2A-2E present Tables 2A-2E. FIG. 2A shows melanoma HLA A*0301 phosphopeptides, A*0101 phosphopeptides, B*4402 phosphopeptides, B*2705 phosphopeptides, and B*1402 phosphopeptides. FIG. 2B shows melanoma and/or leukemia FILA B*0702 phosphopeptides. FIG. 2C shows melanoma HLA A*0301 phosphopeptides, A*0101 phosphopeptides, B*4402 phosphopeptides, B*2705 phosphopeptides, and B*1402 phosphopeptides and their sequence variants. FIG. 2D shows melanoma and/or leukemia HLA B*0702 phosphopeptides and their sequence variants. FIG. 2E shows melanoma HLA-A*0201 phosphopeptides. FIG. 2E shows melanoma HLA-A*0201 phosphopeptides and their sequence variants.

DETAILED DESCRIPTION OF THE INVENTION

[0023] We have identified MHC class I phosphopeptides for use in diagnostics, immunotherapeutics, and adoptive T-cell therapy of melanoma patients. We provide over 200 class I MHC peptides presented on the surface of cancer cells in association with the FILA molecules A*0101 (SEQ ID NO: 70-97), A*0301 (SEQ ID NO: 1-69), and B*4402 (SEQ ID NO: 98-110), B*2705 (SEQ ID NO: 111-162), B*1402 (SEQ ID NO: 163-164), and B*0702 (SEQ ID NO: 165-246). Variants and mimetics of these peptides and of additional class I MHC phosphopeptides are also provided.

[0024] Although individuals in the human population display hundreds of different HLA alleles, some are more prevalent than others. For example, 88% of melanoma patients carry at least one of the six HLA alleles: HLA-A*0201 (29%), HLA-A*0I01 (15%), HLA-A*0301 (14%), HLA-B*4402 (15%), HLA-B*0702 (12%), and HLA-B*-2705 (3%). One of our aims is to provide multiple phosphopeptides presented by each of the six most prevalent alleles and to use them as a cocktail, to optimize coverage of the human population and to minimize the possibility that the tumor will be able to escape immune surveillance by down-regulating expression of any one class I phosphopeptide.

[0025] Phosphopeptides of the invention are not the entire proteins from which they are derived. They are from 8 to 50 contiguous amino acid residues of the native human protein. They contain at least one of the MHC class 1 binding peptides listed in SEQ ID NO: 1-1391. Moreover, at least one of the serine, threonine, or tyrosine residues within the recited sequence is phosphorylated. The phosphorylation may be with a natural phosphorylation (—CH.sub.2—O—PO.sub.3H) or with an enzyme non-degradable, modified phosphorylation, such as (—CH.sub.2—CF.sub.2—PO.sub.3H or —CH.sub.2— CH.sub.2—PO.sub.3H). In certain specified positions, a native amino acid residue in a native human protein may be altered to enhance the binding to the MHC class I molecule. These occur in “anchor” positions of the phosphopeptides, often in positions 1, 2, 3, 9, or 10. Valine, alanine, lysine, leucine tyrosine, arginine, phenylalanine, proline, glutamic acid, threonine, serine, aspartic acid, tryptophan, and methionine may also be used as improved anchoring residues. Anchor residues for different HLA molecules are shown in Table 1, Some phosphopeptides may contain more than one of the peptides listed in SEQ ID NO: 1-1391, for example, if they are overlapping, adjacent, or nearby within the native protein from which they are derived, Phosphopeptides can also be mixed together to form a cocktail. The phosphopeptides may be in an admixture, or they may be linked together in a concatamer as a single molecule. Linkers between individual phosphopeptides may be used; these may, for example, be formed by any 10 to 20 amino acid residues. The linkers may be random sequences, or they may be optimized for degradation by dendritic cells.

TABLE-US-00001 TABLE 1 Optimal anchor residues for HLA molecules HLA A*0201 Residue 2 = L, M Residue 9 or last residue = V HLA A*0301 Residue 2 = L, M, Residue 9 or last residue = K HLA A*0101 Residue 2 = T, S Residue 3 = D, E Residue 9 or last residue = Y HLA B*2705 Residue 1 = R Residue 2 = R Residue 9 or last residue L, F, K, R, M HLA B*0702 Residue 2 = P Residue 9 or last residue = L, M, V, F HLA B*4402 Residue 2 = E Residue 9 or last residue = F, Y, W

[0026] The chemical structure of a phosphopeptide mimetic appropriate for use in the present invention may closely approximate the natural phosphorylated residue which is mimicked, and also be chemically stable (e.g., resistant to dephosphorylation by phosphatase enzymes). This can be achieved with a synthetic molecule in which the phosphorous atom is linked to the amino acid residue, not through oxygen, but through carbon. In one embodiment, a CF2 group links the amino acid to the phosphorous atom. Mimetics of several amino acids which are phosphorylated in nature can be generated by this approach. Mimetics of phosphoserine, phosphothreonine, and phosphotyrosine can be generated by placing a CF2 linkage from the appropriate carbon to the phosphate moiety. The mimetic molecule L-2-amino-4 (diethylphosphono)-4,4-difluorobutanoic acid (F2Pab) may substitute for phosphoserine (Otaka et al., Tetrahedron Letters 36: 927-930 (1995)). L-2-amino-4-phosphono-4,4difluoro-3-methylbutanoic acid (F2Pmb) may substitute for phosphothreonine. L-2-amino-4-phosphono (difluoromethyl) phenylalanine (F2Pmp) may substitute for phosphotyrosine (Akamatsu et al., Bioorg & Med Chem. 5: 157-163 (1997); Smyth et al., Tetrahedron Lett. Tetrahedron Lett. 33, 4137-4140 (1992)).

[0027] Alternatively, the oxygen bridge of the natural amino acid may be replaced with a methylene group.

[0028] Compositions comprising the phosphopeptide are typically substantially free of other human proteins or peptides. They can be made synthetically or by purification from a biological source. They can be made recombinantly. Desirably they are at least 90%, at least 95%, at least 99% pure. For administration to a human body, they do not contain other components that might, be harmful to a human recipient. The compositions are typically devoid of cells, both human and recombinant producing cells. However, as noted below, in some cases, it may be desirable to load dendritic cells with a phosphopeptide and use those loaded dendritic cells as either an immunotherapy agent themselves, or as a reagent to stimulate a patient's T cells ex vivo. The stimulated T cells can be used as an immunotherapy agent. In some cases, It may be desirable to form a complex between a phosphopeptide and an HLA molecule of the appropriate type. Such complexes may be formed in vitro or in vivo. Such complexes are typically tetrameric with respect to an HLA-phosphopeptide complex. Under certain circumstances it may be desirable to add additional proteins or peptides, for example, to make a cocktail having the ability to stimulate an immune response in a number of different HLA type hosts. Alternatively, additional proteins or peptide can provide an interacting function within a single host, such as an adjuvant function or a stabilizing function. As an example, other tumor antigens can be used in admixture with the phosphopeptides, such that multiple different immune responses are induced in a single patient.

[0029] Administration of phosphopeptides to a mammalian recipient may be accomplished using long phosphopeptides, e.g., longer than 15 residues, or using phosphopeptide-loaded dendritic cells. See Melief, J. Med. Sciences 2009; 2:43-45. The immediate goal is to induce activation of CD8.sup.+ T cells. Additional components which can be administered to the same patient, either at the same time or close in time (e.g., within 21 days of each other) include TLR-ligand oligonucleotide CpG and related phosphopeptides that have overlapping sequences of at least 6 amino acid residues. To ensure efficacy, mammalian recipients should express the appropriate human HLA molecules to bind to the phosphopeptides. Transgenic mammals can be used as recipients, for example, if they express appropriate human HLA molecules. If a mammal's own immune system recognizes a similar phosphopeptide then it can be used as model system directly, without introducing a transgene. Useful models and recipients may be at increased risk of developing metastatic cancer, such as metastatic melanoma. Other useful models and recipients may be predisposed, e.g., genetically or environmentally, to develop melanoma or other cancer.

[0030] Phosphopeptide-loaded dendritic cells can also be used to transfuse a cancer patient or a patient at risk of cancer. The composition of dendritic cells can be provided with a single phosphopeptide loaded in the cells. Thus the dendritic cells are homogenous with respect to the loaded phosphopeptide. The homogeneity may not be perfectly achievable. The desired phosphopeptide may be form at least 20%, at least 50%, at least 70%, or at least 90% of the phosphopeptides loaded in the compositions. Additional components may be added to the composition to be administered, such as immune adjuvants, stabilizers, and the like. The particular phosphopeptides were identified on the surfaces of particular cancer cells, but they may be found on other types of cancer cells as well, including but not limited to melanoma, ovarian cancer, breast cancer, colorectal cancer, squamous carcinoma of the lung, sarcoma, renal cell carcinoma, pancreatic carcinomas, squamous tumors of the head and neck, leukemia, brain cancer, liver cancer, prostate cancer, ovarian cancer, and cervical cancer.

[0031] Antibodies and antibody-like molecules containing an antigen-binding region are useful, inter alia, for analyzing tissue to determine the pathological nature of tumor margins. Such tissue may be obtained from a biopsy, for example. Other samples which may be tested include blood, serum, plasma, and lymph. Antibodies to peptides may be generated using methods that are well known in the art. For the production of antibodies, various host animals, including rabbits, mice, rats, goats and other mammals, can be immunized by injection with a peptide. They may be conjugated to carrier proteins such as KLH or tetanus toxoid. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Methods of immunization to achieve a polyclonal antibody response are well known in the art, as are methods for generating hybridomas and monoclonal antibodies.

[0032] For preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies can optionally be produced in germ-free animals (see PCT/US90/02545). Human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). Techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci, U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for desired epitopes together with genes from a human antibody molecule of appropriate biological activity can be used.

[0033] Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e., “humanized” antibodies), single chain (recombinant), Fab fragments, and fragments produced by a Fab expression library. Any of these molecules which contain an antigen binding region specific for a phosphopeptide relative to its cognate non-phosphorylated peptide may be used. These molecules can be used as diagnostic agents for the diagnosis of conditions or diseases (such as cancer) characterized by expression or overexpression of antigen peptides, or in assays to monitor a patient's responsiveness to an anti-cancer therapy. Antibodies specific for one or more of the antigen phosphopeptides can be used as diagnostics for the detection of the antigen phosphopeptides in cancer cells.

[0034] The antibodies or antibody fragments of the present invention can be combined with a carrier or diluent to form a composition. In one embodiment, the carrier is a pharmaceutically acceptable carrier. Such carriers and diluents include sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0035] The antigen phosphopeptides are known to be expressed on a variety of cancer cell types. Thus, they can be used where appropriate, in treating, diagnosing, vaccinating, preventing, retarding, and attenuating melanoma, ovarian cancer, breast cancer, colorectal cancer, squamous carcinoma of the lung, sarcoma, renal cell carcinoma, pancreatic carcinomas, squamous tumors of the head and neck, leukemia, brain cancer, liver cancer, prostate cancer, ovarian cancer, and cervical cancer.

[0036] Antibodies generated with specificity for the antigen phosphopeptides can be used to detect the corresponding phosphopeptides in biological samples. The biological sample could come from an individual who is suspected of having cancer and thus detection would serve to diagnose the cancer. Alternatively, the biological sample may come from an individual known to have cancer, and detection of the antigen phosphopeptides would serve as an indicator of disease prognosis, cancer characterization, or treatment efficacy. Appropriate immunoassays are well known in the art and include, but are not limited to, immunohistochemistry, How cytometry, radioimmunoassay, western blotting, and ELISA. Biological samples suitable for such testing include, but are not limited to, cells, tissue biopsy specimens, whole blood, plasma, serum, sputum, cerebrospinal fluid, pleural fluid, and urine. Antigens recognized by T cells, whether helper T lymphocytes or CTL, are not recognized as intact proteins, but rather as small peptides that associate with class I or class II MHC proteins on the surface of cells. During the course of a naturally occurring immune response antigens that, are recognized in association with class IT MHC molecules on antigen presenting cells are acquired from outside the cell, internalized, and processed into small peptides that associate with the class II MHC molecules. Conversely, the antigens that give rise to proteins that are recognized in association with class I MHC molecules are generally proteins made within the cells, and these antigens are processed and associate with class I MHC molecules. It is now well known that the peptides that associate with a given class I or class II MHC molecule are characterized as having a common binding motif, and the binding motifs for a large number of different class I and II MHC molecules have been determined. It is also well known that synthetic peptides can be made which correspond to the sequence of a given antigen and which contain the binding motif for a given class I or II MHC molecule. These peptides can then be added to appropriate antigen presenting cells, and the antigen presenting cells can be used to stimulate a T helper cell or CTL response either in vitro or in vivo. The binding motifs, methods for synthesizing the peptides, and methods for stimulating a T helper cell or CTL response are all well known and readily available.

[0037] Kits may be composed for help in diagnosis, monitoring, or prognosis. The kits are to facilitate the detecting and/or measuring cancer-specific phosphoproteins. Such kits contain in a single or divided container, a molecule comprising an antigen-binding region. Such molecules are antibodies or antibody-like molecules. Additional components which may be included in the kit include solid supports, detection reagents, secondary antibodies, instructions for practicing, vessels for running assays, gels, control samples, and the like. The antibody or antibody-like molecules may be directly labeled, as an option.

[0038] The antigens of this invention may take the form of antigen peptides added to autologous dendritic cells and used to stimulate a T helper cell or CTL response in vitro. The in vitro generated T helper cells or CTL can then be infused into a patient with cancer (Yee et al., 2002), and specifically a patient with a form of cancer that expresses one or more of antigen phosphopeptides. The antigen phosphopeptides may also be used to vaccinate an individual. The antigen phosphopeptides may be injected alone, but most often they would be administered in combination with an adjuvant. The phosphopeptides may also be added to dendritic cells in vitro, with the loaded dendritic cells being subsequently transferred into an individual with cancer in order to stimulate an immune response. Alternatively, the loaded dendritic cells may be used to stimulate CD8.sup.+ T cells ex vivo with subsequent reintroduction of the stimulated T cells to the patient. Although a particular phosphopeptide may be identified on a particular cancer cell type, it may be found on other cancer cell types. Thus a particular phosphopeptide may have use for treating and vaccinating against multiple cancer types.

[0039] Phosphopeptide analogs can readily be synthesized that retain their ability to stimulate a particular immune response, but which also gain one or more beneficial features, such as those described below. [0040] a. Substitutions may be made in the phosphopeptide at residues known to interact with the MHC molecule. Such substitutions can have the effect of increasing the binding affinity of the phosphopeptide for the MHC molecule and can also increase the half-life of the phosphopeptide-MHC complex, the consequence of which is that the analog is a more potent stimulator of an immune response than is the original peptide. [0041] b. Additionally, the substitutions may have no effect on the immunogenicity of the phosphopeptide per se, but rather than may prolong its biological half-life or prevent it from undergoing spontaneous alterations which might otherwise negatively impact on the immunogenicity of the peptide.

[0042] The antigen phosphopeptides of this invention can also be used as a vaccine for cancer, and more specifically for melanoma, leukemia, ovarian, breast, colorectal, or lung squamous cancer, sarcoma, renal cell carcinoma, pancreatic carcinomas, squamous tumors of the head and neck, brain cancer, liver cancer, prostate cancer, ovarian cancer, and cervical cancer. The antigens may take the form of phosphoproteins, or phosphopeptides. The vaccine may include only the antigens of this invention or they may include other cancer antigens that have been identified. Pharmaceutical carriers, diluents and excipients are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used. The vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.

[0043] The composition may be administered parenterally, either systemically or topically. Parenteral routes include subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. One or more such routes may be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.

[0044] It is understood that a suitable dosage of an immunogen will depend upon the age, sex, health, and weight of the recipient, the kind of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired, however, the most preferred dosage can be tailored to the individual subject, as determined by the researcher or clinician. The total dose required for any given treatment will commonly be determined with respect to a standard reference dose based on the experience of the researcher or clinician, such dose being administered either in a single treatment or in a series of doses, the success of which will depend on the production of a desired immunological result (i.e., successful production of a T helper cell and/or CTL-mediated response to the antigen, which response gives rise to the prevention and/or treatment desired). Thus, the overall administration schedule must be considered in determining the success of a course of treatment and not whether a single dose, given in isolation, would or would not produce the desired immunologically therapeutic result or effect. Thus, the therapeutically effective amount (i.e., that producing the desired T helper cell and/or CTL-mediated response) will depend on the antigenic composition of the vaccine used, the nature of the disease condition, the severity of the disease condition, the extent of any need to prevent such a condition where it has not already been detected, the manner of administration dictated by the situation requiring such administration, the weight and state of health of the individual receiving such administration, and the sound judgment of the clinician or researcher. Needless to say, the efficacy of administering additional doses, and of increasing or decreasing the interval, may be re-evaluated on a continuing basis, in view of the recipient's immunocompetence (for example, the level of T helper cell and/or CTL activity with respect to tumor-associated or tumor-specific antigens).

[0045] The concentration of the T helper or CTL stimulatory peptides of the invention in pharmaceutical formulations are subject to wade variation, including anywhere from less than 0.01% by weight to as much as 50% or more. Factors such as volume and viscosity of the resulting composition should also be considered. The solvents, or diluents, used for such compositions include water, possibly PBS (phosphate buffered saline), or saline itself, or other possible carriers or excipients. The immunogens of the present invention may also be contained in artificially created structures such as liposomes, which structures may or may not contain additional molecules, such as proteins or polysaccharides, inserted in the outer membranes of said structures and having the effect of targeting the liposomes to particular areas of the body, or to particular cells within a given organ or tissue. Such targeting molecules may commonly be some type of immunoglobulin. Antibodies may work particularly well for targeting the liposomes to tumor cells.

[0046] The vaccine compositions may be used prophylactically for the purposes of preventing, reducing the risk of, delaying initiation of a cancer in an individual that does not currently have cancer. Or they may be used to treat an individual that already has cancer, so that recurrence or metastasis is delayed or prevented. Prevention relates to a process of prophylaxis in which the individual is immunized prior to the induction or onset of cancer. For example, individuals with a history of severe sunburn and at risk for developing melanoma, might be immunized prior to the onset of the disease. Alternatively, individuals that already have cancer can be immunized with the antigens of the present invention so as to stimulate an immune response that would be reactive against the cancer. A clinically relevant immune response would be one in which the cancer partially or completely regresses and is eliminated from the patient, and it would also include those responses in which the progression of the cancer is blocked without being eliminated. Similarly, prevention need not be total, but may result in a reduced risk, delayed onset, or delayed progression or metastasis.

[0047] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

Example 1

[0048] The present example encompasses inter alia a set of phosphorylated peptides presented by HLA A*0101, A*0301 and B*4402 on the surface of melanoma cells that have the potential to (a) stimulate an immune response to the cancer, (b) to function as immunotherapeutics in adoptive T-cell therapy or as a vaccine, (c) to facilitate antibody recognition of the tumor boundaries in surgical pathology samples, and (d) act as biomarkers for early detection of the disease. The present invention provides at least 246 class I MHC peptides presented on the surface of melanoma, cells in association with the HLA molecules A*0101, A*0301, and B*4402.

[0049] Tables 2A through 2E, are shown in FIG. 2A-2E. Sequence identifiers are listed in the first column. UniProt database sequences provide the sequences of the full human proteins from which the peptides are derived. The UniProt sequences are incorporated by reference.

[0050] The class I phosphopeptide antigens reported here allow adoptive T-cell therapy to be extended to melanoma patients that do not express the HLA-A*0201 allele and also make it possible to treat a, variety of other cancers by the same approach.

[0051] We have also shown that we can clone the T-cell receptor on the murine cytotoxic T-cells and then inject the corresponding DNA into normal human T-cells. This process turns them into cytotoxic T-cells that now recognize cancer cells that express the same class I phosphopeptides derived from IRS-2 and β-catenin. In short, we have now demonstrated that this process can be used to convert cancer patient T-cells into activated cytotoxic T-cell that recognize class I phosphopeptides and kill their tumor. These experiments also open the door for using class I phosphopeptides in adopted T-cell therapy of cancer. This approach has shown dramatic success in the treatment, of advanced stage metastatic melanoma. In conclusion, it should be noted that HLA A*0201 and HLA *A0301 both present peptides from the IRS-2 protein that contain the same phosphorylation site, Seri 100. RVApSPTSGV (SEQ ID NO: 1289) binds to HLA A*0201 and both RVApSPTSGVK (SEQ ID NO: 53) and RVApSPTSGVKR (SEQ ID NO: 54) bind to HLA A*0301. Neither of the A*0301 peptides bind to A*0201 and the A*0201 peptide cannot be presented by the A*0301 molecule.

REFERENCES

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