Population-based immunogenic peptide identification platform

Abstract

The disclosure relates to methods of identifying fragments of a polypeptide that are immunogenic for a specific human subject, methods of preparing pharmaceutical compositions comprising such polypeptide fragments, pharmaceutical compositions comprising such polypeptide fragments, and methods of treatment using such compositions. The methods comprise identifying a fragment of the polypeptide that binds to multiple HLA of individual subjects.

What is claimed is: 
     
         1 . A pharmaceutical composition for treatment of a disease or disorder in a subject of a target human population, comprising one or more polypeptides, each comprising at least a first region and a second region,
 (a) the first region being of 10-50 amino acids in length comprising a first amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and   (b) the second region being of 10-50 amino acids in length comprising a second amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population;   wherein the amino acid sequence of the T cell epitope of each of first and second regions comprise different sequences.   
     
     
         2 . The pharmaceutical composition of  claim 1 , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides. 
     
     
         3 . The pharmaceutical composition of  claim 1 , comprising 2-40 different polypeptides. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein the T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules of at least 10% of subjects in the target population comprises 13 to 17 amino acids. 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the first region of 10-50 amino acids in length is from an antigen; and the second region of 10-50 amino acids in length is from a same or different antigen. 
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein the epitopes of the first and second regions are from a single antigen. 
     
     
         7 . The pharmaceutical composition of  claim 1 , wherein the epitopes of the first and second regions are from two or more different antigens. 
     
     
         8 . The pharmaceutical composition of  claim 5 , wherein the antigen is a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. 
     
     
         9 . The pharmaceutical composition of  claim 5 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         10 . The pharmaceutical composition of  claim 6 , wherein the two or more different antigens are selected from the antigens listed in Tables 2 to 7 and/or different cancer associated antigens. 
     
     
         11 . The pharmaceutical composition of  claim 9 , wherein one or more of the antigens are cancer testis antigens (CTAs). 
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein the one or more polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in a corresponding antigen. 
     
     
         13 . The pharmaceutical composition of  claim 1 , wherein the one or more polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the first region and second region and that
 (i) corresponds to a fragment of a human polypeptide expressed in healthy cells;   (ii) is a T cell epitope capable of binding to at least three HLA class I molecules of at least 10% of subjects in the target population; or   (iii) meets both requirements (i) and (ii).   
     
     
         14 . The pharmaceutical composition of  claim 1 , wherein the target population is cancer patients and wherein each of the first region and second region comprises an amino acid sequence that is an HLA class I-binding T cell epitope, and wherein for each T cell epitope,
 (i) at least 10% of subjects in the target population express a tumor associated antigen selected from the antigens listed in Table 2 that comprises the T cell epitope; and   (ii) at least 10% of subjects in the target population have at least three HLA class I molecules capable of binding to the T cell epitope;   wherein the T cell epitope of the first and second regions are different from each other.   
     
     
         15 . The pharmaceutical composition of  claim 1 , further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. 
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,  bacillus  Calmette-Guerin (BCG), corynbacterium  parvum , levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. 
     
     
         17 . A kit comprising, one or more separate containers each container comprising:
 (i) one or more polypeptides comprising at least a first region and a second region,
 (a) the first region of 10-50 amino acids in length comprising a first amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and 
 (b) the second region of 10-50 amino acids in length comprising a second amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; wherein the amino acid sequence of the T cell epitope of each of first and second regions comprise different sequences and 
   (ii) a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof.   
     
     
         18 . The kit of  claim 17 , further comprising a package insert. 
     
     
         19 . A pharmaceutical composition comprising: one or more nucleic acid molecules expressing one or more polypeptides comprising at least a first region and a second region,
 (a) the first region of 10-50 amino acids in length comprising a first amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and   (b) the second region of 10-50 amino acids in length comprising a second amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; wherein the amino acid sequence of the T cell epitope of each of first and second regions comprise different sequences.   
     
     
         20 . A method of preparing a polypeptide, or a polynucleic acid that encodes a polypeptide, for use in a method of inducing an immune response in a subject of a target human population, the method comprising:
 (i) selecting:
 (a) a relevant model human population comprising a plurality of subjects each defined by HLA class I genotype and/or by HLA class II genotype; or 
 (b) one relevant model human population comprising a plurality of subjects each defined by HLA class I genotype and one relevant model human population comprising a plurality of subjects each defined by HLA class II genotype; 
   (ii) identifying a fragment of up to 50 consecutive amino acids of an antigen that comprises:
 (a) a T cell epitope capable, in a high percentage of subjects of the model population selected in step (i) that is defined by HLA class I genotype, of binding to at least three HLA class I molecules of individual subjects of the model population; 
 (b) a T cell epitope capable, in a high percentage of subjects of the model population selected in step (i) that is defined by HLA class II genotype, of binding to at least three HLA class II molecules of individual subjects of the model population; or 
 (c) a T cell epitope capable, in a high percentage of subjects of the model population selected in step (i) that is defined by HLA class I genotype, of binding to at least three HLA class I molecules of individual subjects of the model population and a T cell epitope capable, in a high percentage of subjects of the model population selected in step (i) that is defined by HLA class II genotype, of binding to at least three HLA class II molecules of individual subjects of the model population; and 
   (iii) preparing a polypeptide, or a polynucleic acid that encodes a polypeptide that comprises one or more fragments identified in step (ii).   
     
     
         21 . The method of  claim 20 , further comprising prior to step (iii), selecting a longer fragment of the antigen if the fragment selected in step (ii) is an HLA class I—binding epitope, which longer fragment comprises an amino acid sequence that
 (a) comprises the fragment selected in step (ii); and 
 (b) is an HLA class II molecule-binding T cell epitope capable, in a high percentage of subjects of the model population selected in step (i) that is defined by HLA class II genotype, of binding to at least three, or the most possible HLA class II molecules of individual subjects of the model population. 
 
     
     
         22 . The method of  claim 20 , further comprising prior to step (iii), repeating steps (i) to (ii) to identify on or more additional amino acid sequences of up to 50 consecutive amino acids of the same or a different polypeptide to the first amino acid sequence. 
     
     
         23 . A method of inducing an immune response in a subject of a target human population, the method comprising,
 administering to the subject a pharmaceutical composition comprising one or more polypeptides comprising at least a first region and a second region,   (a) the first region being of 10-50 amino acids in length comprising a first amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and   (b) the second region being of 10-50 amino acids in length comprising a second amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population;   wherein the amino acid sequence of the T cell epitope of each of first and second regions comprise different sequences.   
     
     
         24 . The method of  claim 23 , further comprising prior to the administering step, determining if the subject is likely to have an have a clinical response to administration of a pharmaceutical composition by
 (i) assaying a biological sample of the subject to determine HLA genotype of the subject;   (ii) determining that the pharmaceutical composition comprises two or more sequences that are a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and   (iii) determining the probability that a tumor of the subject expresses one or more antigen corresponding to the T cell epitopes identified in step (ii) using population expression data for each antigen, to identify the likelihood of the subject to have a clinical response to administration of the pharmaceutical composition.   
     
     
         25 . The method of  claim 23 , wherein the first region of 10-50 amino acids in length is from an antigen; and the second region of 10-50 amino acids in length is from a same or different antigen. 
     
     
         26 . The method of  claim 23 , wherein the epitopes of the first and second regions are from two or more different antigens. 
     
     
         27 . The method of  claim 25 , wherein the antigen is a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. 
     
     
         28 . The method of  claim 23 , wherein the T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules of at least 10% of subjects in the target population comprises 13 to 17 amino acids. 
     
     
         29 . A pharmaceutical composition for treatment of a disease or disorder in a subject of a target human population, comprising
 (a) at least two polypeptides, each of the at least two polypeptides being 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population, and/or at least three HLA class II molecules of at least 10% of subjects in the target population, wherein the amino acid sequence of the T cell epitope of each of the at least two polypeptides are different from each other; and   (b) a pharmaceutically-acceptable adjuvant.   
     
     
         30 . The pharmaceutical composition of  claim 29 , comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides. 
     
     
         31 . The pharmaceutical composition of  claim 29 , comprising 3-40 different polypeptides. 
     
     
         32 . The pharmaceutical composition of  claim 29 , wherein the T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules of at least 10% of subjects in the target population comprises 13 to 17 amino acids. 
     
     
         33 . The pharmaceutical composition of  claim 29 , wherein the epitopes of the amino acid sequences of the at least two polypeptides are from a single antigen. 
     
     
         34 . The pharmaceutical composition of  claim 29 , wherein the epitopes of the amino acid sequences of the at least two polypeptides are from two or more different antigens. 
     
     
         35 . The pharmaceutical composition of  claim 33 , wherein the antigen is a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. 
     
     
         36 . The pharmaceutical composition of  claim 33 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         37 . The pharmaceutical composition of  claim 34 , wherein the two or more different antigens are selected from the antigens listed in Tables 2 to 7 and/or different cancer associated antigens. 
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein one or more of the antigens are cancer testis antigens (CTAs). 
     
     
         39 . The pharmaceutical composition of  claim 29 , wherein each of the at least two polypeptides being 10-50 amino acids in length is from an antigen a same or different antigen. 
     
     
         40 . The pharmaceutical composition of  claim 29 , wherein the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in a corresponding antigen. 
     
     
         41 . The pharmaceutical composition of  claim 29 , wherein two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         42 . The pharmaceutical composition of  claim 41 , comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. 
     
     
         43 . The pharmaceutical composition of  claim 42 , wherein the joined polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two polypeptides and that
 (i) corresponds to a fragment of a human polypeptide expressed in healthy cells;   (ii) is a T cell epitope capable of binding to at least three HLA class I molecules of at least 10% of subjects in the target population; or   (iii) meets both requirements (i) and (ii).   
     
     
         44 . The pharmaceutical composition of  claim 29 , wherein the target population is cancer patients and wherein each polypeptide comprises an amino acid sequence that is an HLA class I-binding T cell epitope, and wherein for each T cell epitope,
 (i) at least 10% of subjects in the target population express a tumor associated antigen selected from the antigens listed in Table 2 that comprises the T cell epitope; and   (ii) at least 10% of subjects in the target population have at least three HLA class I molecules capable of binding to the T cell epitope;   wherein the T cell epitope of the at least two polypeptides are different from each other.   
     
     
         45 . The pharmaceutical composition of  claim 29 , further comprising a pharmaceutically acceptable diluent, carrier, preservative, or combination thereof. 
     
     
         46 . The pharmaceutical composition of  claim 29 , wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,  bacillus  Calmette-Guerin (BCG), corynbacterium  parvum , levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. 
     
     
         47 . A pharmaceutical composition for treatment of a disease or disorder in a subject of a target human population, comprising
 (a) a polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and   (b) a pharmaceutically-acceptable adjuvant.   
     
     
         48 . The pharmaceutical composition of  claim 47 , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, each of the different polypeptides being 10-50 amino acids in length comprising a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. 
     
     
         49 . The pharmaceutical composition of  claim 48 , comprising 2-40 different polypeptides. 
     
     
         50 . The pharmaceutical composition of  claim 47 , wherein the T cell epitope that binds at least three HLA class I molecules of the subject comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules comprises 13 to 17 amino acids. 
     
     
         51 . The pharmaceutical composition of  claim 48 , comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from a single antigen. 
     
     
         52 . The pharmaceutical composition of  claim 48 , comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from two or more different antigens. 
     
     
         53 . The pharmaceutical composition of  claim 51 , wherein the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen. 
     
     
         54 . The pharmaceutical composition of  claim 51 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         55 . The pharmaceutical composition of  claim 51 , comprising at least two different polypeptides, wherein two of the polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         56 . The pharmaceutical composition of  claim 47 , wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,  bacillus  Calmette-Guerin (BCG), corynbacterium  parvum , levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. 
     
     
         57 . The pharmaceutical composition of  claim 47 , comprising at least two different polypeptides, wherein two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         58 . The pharmaceutical composition of  claim 57 , comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. 
     
     
         59 . The pharmaceutical composition of  claim 58 , wherein the joined polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two polypeptides and that
 (i) corresponds to a fragment of a human polypeptide expressed in healthy cells of the subject;   (ii) is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or   (iii) meets both requirements (i) and (ii).   
     
     
         60 . The pharmaceutical composition of  claim 48 , wherein the at least two polypeptides do not comprise any amino acid sequences that
 (i) correspond to a fragment of a human polypeptide expressed in healthy cells; or   (ii) correspond to a fragment of a human polypeptide expressed in healthy cells and is a T cell epitope capable of binding to at least two HLA class I molecules of the subject.   
     
     
         61 . A method of identifying and treating a subject of a target population of cancer patients who will likely have a clinical response to administration of a pharmaceutical composition according to  claim 1 , the method comprising,
 (i) assaying a biological sample of the subject to determine HLA genotype of the subject;   (ii) determining that the pharmaceutical composition comprises two or more sequences that are a T cell epitope capable of binding to at least three HLA class I molecules of the subject;   (iii) determining the probability that a tumor of the subject expresses one or more antigen corresponding to the T cell epitopes identified in step (ii) using population expression data for each antigen, to identify the likelihood of the subject to have a clinical response to administration of the pharmaceutical composition; and   (iv) administering the composition of  claim 1  to the identified subject.   
     
     
         62 . The method of  claim 61 , further comprising prior to the administering step
 assaying a tumor sample from the subject to determine that the three or more peptides of the pharmaceutical composition comprise two or more different amino acid sequences each of which is
 a. a fragment of a cancer-associated antigen expressed by cancer cells of the subject as determined in step (i); and 
 b. a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and 
   confirming the subject as likely to have a clinical response to the method of treatment.   
     
     
         63 . The method of  claim 61 , wherein the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides. 
     
     
         64 . The method of  claim 61 , wherein the composition comprises 2-40 different polypeptides. 
     
     
         65 . The method of  claim 61 , wherein the T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population comprises 7 to 11 amino acids, and/or the T cell epitope that binds at least three HLA class II molecules of at least 10% of subjects in the target population comprises 13 to 17 amino acids. 
     
     
         66 . The method of  claim 61 , wherein the first region of 10-50 amino acids in length is from an antigen; and the second region of 10-50 amino acids in length is from a same or different antigen. 
     
     
         67 . The method of  claim 61 , wherein the epitopes of the first and second regions are from a single antigen. 
     
     
         68 . The method of  claim 61 , wherein the epitopes of the first and second regions are from two or more different antigens. 
     
     
         69 . The method of  claim 67 , wherein the antigen is a cancer-associated antigen or a tumor-associated antigen. 
     
     
         70 . The method of  claim 67 , wherein the antigen is selected from the antigens listed in Table 2. 
     
     
         71 . The method of  claim 67 , wherein the two or more different antigens are selected from the antigens listed in Table 2 and/or different cancer associated antigens. 
     
     
         72 . The method of  claim 71 , wherein one or more of the antigens are cancer testis antigens (CTAs). 
     
     
         73 . The method of  claim 61 , wherein the one or more polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are not part of a consecutive sequence flanking the epitope in a corresponding antigen. 
     
     
         74 . The method of  claim 61 , wherein the one or more polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the first region and second region and that
 (i) corresponds to a fragment of a human polypeptide expressed in healthy cells;   (ii) is a T cell epitope capable of binding to at least three HLA class I molecules of at least 10% of subjects in the target population; or   (iii) meets both requirements (i) and (ii).   
     
     
         75 . The method of  claim 61 , wherein the target population is cancer patients and wherein each of the first region and second region comprises an amino acid sequence that is an HLA class I-binding T cell epitope, and wherein for each T cell epitope,
 (iii) at least 10% of subjects in the target population express a tumor associated antigen selected from the antigens listed in Table 2 that comprises the T cell epitope; and   (iv) at least 10% of subjects in the target population have at least three HLA class I molecules capable of binding to the T cell epitope;   
       wherein the T cell epitope of the first and second regions are different from each other. 
     
     
         76 . The method of  claim 61 , wherein the composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. 
     
     
         77 . The method of  claim 61 , wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide,  bacillus  Calmette-Guerin (BCG), corynbacterium  parvum , levamisole, azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete), mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinations thereof. 
     
     
         78 . A kit comprising:
 (a) a first composition comprising (i) a first polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and (ii) a pharmaceutically-acceptable adjuvant;   (b) a second composition comprising (i) a second polypeptide of 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population; and (ii) a pharmaceutically-acceptable adjuvant,   wherein the first and second polypeptides comprise different T cell epitopes.   
     
     
         79 . The kit of  claim 78 , wherein the first composition and/or the second composition comprise one or more additional polypeptides, wherein each additional polypeptide being of 10-50 amino acids in length comprising an amino acid sequence that is a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population, wherein the amino acid sequences comprise different T cell epitopes. 
     
     
         80 . A method of identifying and treating a subject of a target population of cancer patients who will likely have an immune response to administration of a pharmaceutical composition according to  claim 1 , the method comprising,
 (i) assaying a biological sample of the subject to determine HLA genotype of the subject;   (ii) determining that the pharmaceutical composition comprises two or more sequences that are a T cell epitope capable of binding to at least three HLA class I molecules of the subject;   (iii) administering the composition of  claim 1  to the identified subject.   
     
     
         81 . A pharmaceutical composition comprising: a nucleic acid molecule expressing two or more polypeptides, each polypeptide being 10-50 amino acids in length comprising a T cell epitope that binds at least three HLA class I molecules of at least 10% of subjects in the target population and/or at least three HLA class II molecules of at least 10% of subjects in the target population, wherein each of the two or more polypeptides comprises a different T cell epitope, wherein the polypeptides do not comprise amino acid sequences that are adjacent to each other in a corresponding antigen.