Personalised 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 personalised pharmaceutical compositions comprising such polypeptide fragments, human subject-specific 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 the subject.

What is claimed is: 
     
         1 . A human subject-specific pharmaceutical composition for treatment of a disease or disorder in a specific human subject, comprising
 (a) at least two different polypeptides, each of the at least two different polypeptides being 10-50 amino acids in length and comprising a T cell epitope that binds at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, and wherein the T cell epitope of each of the at least two polypeptides are different from each other; and   (b) a pharmaceutically-acceptable adjuvant.   
     
     
         2 . The human subject-specific pharmaceutical composition of  claim 1 , 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. 
     
     
         3 . The human subject-specific pharmaceutical composition of  claim 1 , comprising 3-40 different polypeptides. 
     
     
         4 . The human subject-specific pharmaceutical composition of  claim 1 , 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. 
     
     
         5 . The human subject-specific pharmaceutical composition of  claim 1 , wherein the epitopes of the at least two different polypeptides are from a single antigen. 
     
     
         6 . The human subject-specific pharmaceutical composition of  claim 1 , wherein the epitopes of the at least two different polypeptides are from two or more different antigens. 
     
     
         7 . The human subject-specific pharmaceutical composition of  claim 5 , 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. 
     
     
         8 . The human subject-specific pharmaceutical composition of  claim 7 , wherein the cancer cell is from the subject. 
     
     
         9 . The human subject-specific pharmaceutical composition of  claim 5 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         10 . The human subject-specific pharmaceutical composition of  claim 1 , wherein the at least two different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are part of a consecutive sequence flanking the epitope in a corresponding antigen. 
     
     
         11 . The human subject-specific pharmaceutical composition of  claim 1 , 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. 
     
     
         12 . The human subject-specific pharmaceutical composition of  claim 1 , wherein two of the at least two polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         13 . The human subject-specific pharmaceutical composition of  claim 12 , comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. 
     
     
         14 . The human subject-specific pharmaceutical composition of  claim 13 , 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).   
     
     
         15 . The human subject-specific pharmaceutical composition of  claim 1 , 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.   
     
     
         16 . The human subject-specific pharmaceutical composition of  claim 1 , further comprising a pharmaceutically acceptable diluent, carrier, preservative, or combination thereof. 
     
     
         17 . The human subject-specific pharmaceutical composition of  claim 1 , 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. 
     
     
         18 . A kit comprising, one or more separate containers each container comprising:
 (i) one or more 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 the subject and/or at least three HLA class II molecules of the subject; and   (ii) a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof.   
     
     
         19 . The kit of  claim 18 , 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, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. 
     
     
         20 . The kit of  claim 18 , further comprising a package insert. 
     
     
         21 . A human subject-specific 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 the subject and/or at least three HLA class II molecules of the subject, 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.   
     
     
         22 . The pharmaceutical composition of  claim 21 , wherein the nucleic acid molecule expresses 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 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 the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. 
     
     
         23 . A human subject-specific pharmaceutical composition for treatment of a disease or disorder in a specific human subject, comprising at least one different polypeptides, each of the at least one different polypeptides comprising at least a first region and a second region,
 (i) the first region 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 the subject and/or at least three HLA class II molecules of the subject,   (ii) the second region 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 the subject and/or at least two HLA class II molecules of the subject,
 wherein the amino acid sequence of the T cell epitope of each of the first and second regions of each of the at least three different polypeptides comprise different sequences. 
   
     
     
         24 . The human subject-specific pharmaceutical composition of  claim 23 , 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. 
     
     
         25 . The human subject-specific pharmaceutical composition of  claim 23 , comprising 2-40 different polypeptides. 
     
     
         26 . The human subject-specific pharmaceutical composition of  claim 23 , 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. 
     
     
         27 . The human subject-specific pharmaceutical composition of  claim 23 , wherein the epitopes of the first and second regions are from a single antigen. 
     
     
         28 . The human subject-specific pharmaceutical composition of  claim 23 , wherein the epitopes of the first and second regions are from two or more different antigens. 
     
     
         29 . The human subject-specific pharmaceutical composition of  claim 27 , 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. 
     
     
         30 . The human subject-specific pharmaceutical composition of  claim 29 , wherein the cancer cell is from the subject. 
     
     
         31 . The human subject-specific pharmaceutical composition of  claim 27 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         32 . The human subject-specific pharmaceutical composition of  claim 23 , wherein the polypeptides have been screened to eliminate substantially all neoepitopes that span a junction between the two regions 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).   
     
     
         33 . The human subject-specific pharmaceutical composition of  claim 23 , wherein the at least one 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.   
     
     
         34 . The human subject-specific pharmaceutical composition of  claim 23 , further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. 
     
     
         35 . The human subject-specific pharmaceutical composition of  claim 34 , 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. 
     
     
         36 . A method of preparing a human subject-specific pharmaceutical composition for use in a method of treatment of a specific human subject, the method comprising:
 (j) selecting a fragment of a polypeptide, which fragment has been identified as immunogenic for the subject by
 a) determining whether the fragment comprises:
 1) an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or 
 2) an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or 
 3) or meets both requirements (1) and (2); and 
 
 b) identifying the sequence as a fragment of the polypeptide that is immunogenic for the subject; 
   (ii) selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, which consecutive amino acids comprise the amino acid sequence of the fragment selected in step (i); and   (iii) preparing a subject-specific pharmaceutical composition having as active ingredients one or more polypeptides that together have all of the amino acid sequences selected in the preceding steps.   
     
     
         37 . The method of  claim 36 , further comprising prior to the preparing step repeating steps (i) to (ii) to select a second amino acid sequence of up to 50 consecutive amino acids of the same or a different polypeptide to the first amino acid sequence. 
     
     
         38 . The method of  claim 37 , further comprising, further repeating prior to the preparing step, steps (i) to (ii) one or more times to select one or more additional amino acid sequences of up to 50 consecutive amino acids of the same or different polypeptides to the first and second amino acid sequences. 
     
     
         39 . The method of  claim 36 , further comprising prior to the preparing step selecting a longer fragment of the polypeptide if the fragment selected in step (i) is an HLA class I-binding epitope, which longer fragment comprises the fragment selected in step (i); and is a T cell epitope capable of binding at least three HLA class II molecules of the subject. 
     
     
         40 . The method of  claim 36 , wherein each polypeptide either consists of one of the selected amino acid sequences, or comprises or consists of two or more of the selected amino acid sequences arranged end to end or overlapping in a single joined polypeptide. 
     
     
         41 . The method of  claim 36 , wherein any neoepitopes formed at the junction between any two of the selected amino acid sequences arranged end to end in a single joined polypeptide have been screened to eliminate substantially all polypeptides comprising a neoepitope amino acid sequence 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 two HLA class I molecules of the subject; or   (iii) meets both requirements (i) and (ii).   
     
     
         42 . The method of  claim 36 , wherein the one or more polypeptides have been screened to eliminate polypeptides comprising an amino acid sequence that
 (i) corresponds to a fragment of a human polypeptide expressed in healthy cells; or   (ii) corresponds 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.   
     
     
         43 . The method of  claim 36 , further comprising determining HLA class I genotype and HLA class II genotype from a biological sample of the subject prior to step (i). 
     
     
         44 . The method of  claim 43 , wherein the determining HLA class I genotype and HLA class II genotype is performed by sequence based typing (SBT) methods. 
     
     
         45 . The method of  claim 43 , wherein the determining HLA class I genotype and HLA class II genotype is performed by sequencing, next generation sequencing, sequence specific primer (SSP) methods, or sequence specific oligonucleotide (SSO) methods. 
     
     
         46 . The method of  claim 43 , wherein the biological sample is blood, serum, plasma, saliva, buccal swab, urine, expiration, cell, or tissue. 
     
     
         47 . The method of  claim 43 , wherein the biological sample is saliva or a buccal swab. 
     
     
         48 . A method of treating a cancer in a specific human subject in need thereof comprising,
 administering to a specific human subject a pharmaceutical composition comprising at least one polypeptide,   each of the at least one polypeptide being 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 the subject and/or at least three HLA class II molecules of the subject,   wherein the T cell epitope of each of the at least one polypeptide is from an antigen that is specific for the cancer.   
     
     
         49 . The method of  claim 48 , 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, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other, and are from one or more antigens that are expressed by a cancer cell from the subject. 
     
     
         50 . The method of  claim 48 , wherein the composition comprises 2-40 different polypeptides. 
     
     
         51 . The method of  claim 48 , wherein 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. 
     
     
         52 . The method of  claim 48 , wherein the composition comprises at least two different polypeptides and the epitopes of the amino acid sequences of the at least two different polypeptides are from a single antigen. 
     
     
         53 . The method of  claim 48 , wherein the composition comprises at least two different polypeptides and the epitopes of the at least two different polypeptides are from two or more different antigens. 
     
     
         54 . The method of  claim 48 , wherein the one or more antigen is a neoantigen expressed by a cancer cell, a cancer-associated antigen, or a tumor-associated antigen. 
     
     
         55 . The method of  claim 48 , wherein the one or more antigen is selected from the antigens listed in Table 2. 
     
     
         56 . The method of  claim 48 , wherein the at least one different polypeptides further comprise up to 10 amino acids flanking the T cell epitope that are part of a consecutive sequence flanking the epitope in a corresponding antigen. 
     
     
         57 . The method of  claim 48 , wherein the at least one 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. 
     
     
         58 . The method of  claim 48 , wherein the composition comprises at least two different polypeptides and two of the polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         59 . The method of  claim 58 , comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. 
     
     
         60 . The method of  claim 59 , 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).   
     
     
         61 . The method of  claim 48 , wherein the at least one polypeptide does 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.   
     
     
         62 . The method of  claim 48 , wherein the composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof. 
     
     
         63 . The method of  claim 62 , 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. 
     
     
         64 . The method of  claim 48 , further comprising administering a chemotherapeutic agent, a targeted therapy, radiation therapy, a checkpoint inhibitor, another immunotherapy, or combination thereof. 
     
     
         65 . A human subject-specific pharmaceutical composition for treatment of a disease or disorder in a specific human subject, 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 the subject and/or at least three HLA class II molecules of the subject; and (b) a pharmaceutically-acceptable adjuvant. 
     
     
         66 . The human subject-specific pharmaceutical composition of  claim 65 , 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 the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T cell epitope of each of the different polypeptides are different from each other. 
     
     
         67 . The human subject-specific pharmaceutical composition of  claim 66 , comprising 2-40 different polypeptides. 
     
     
         68 . The human subject-specific pharmaceutical composition of  claim 65 , 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. 
     
     
         69 . The human subject-specific pharmaceutical composition of  claim 66 , comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from a single antigen. 
     
     
         70 . The human subject-specific pharmaceutical composition of  claim 66 , comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are from two or more different antigens. 
     
     
         71 . The human subject-specific pharmaceutical composition of  claim 69 , 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. 
     
     
         72 . The human subject-specific pharmaceutical composition of  claim 71 , wherein the cancer cell is from the subject. 
     
     
         73 . The human subject-specific pharmaceutical composition of  claim 69 , wherein the antigen is selected from the antigens listed in Tables 2 to 7. 
     
     
         74 . The human subject-specific pharmaceutical composition of  claim 69 , comprising at least two different polypeptides, wherein two of the polypeptides are arranged end to end or overlapping in a joined polypeptide. 
     
     
         75 . The human subject-specific pharmaceutical composition of  claim 65 , 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. 
     
     
         76 . The human subject-specific pharmaceutical composition of  claim 65 , 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. 
     
     
         77 . The human subject-specific pharmaceutical composition of  claim 76 , comprising two or more different joined polypeptides, wherein the two or more different joined polypeptides comprise different epitopes from each other. 
     
     
         78 . The human subject-specific pharmaceutical composition of  claim 77 , 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).   
     
     
         79 . The human subject-specific pharmaceutical composition of  claim 66 , 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.   
     
     
         80 . A kit comprising:
 (a) a first human subject-specific pharmaceutical 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 the subject and/or at least three HLA class II molecules of the subject; and (ii) a pharmaceutically-acceptable adjuvant;   (b) a second human subject-specific pharmaceutical 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 the subject and/or at least three HLA class II molecules of the subject; and (ii) a pharmaceutically-acceptable adjuvant,   wherein the first and second polypeptides comprise different T cell epitopes.   
     
     
         81 . The kit of  claim 80 , 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 the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequences comprise different T cell epitopes.