Compositions and Methods of Identifying Tumor Specific Neoantigens

Abstract

The present invention related to immunotherapeutic peptides and their use in immunotherapy, in particular the immunotherapy of cancer. Specifically, the invention provides a method of identifying tumor specific neoantigens that alone or in combination with other tumor-associated peptides serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor responses.

1 .- 20 . (canceled) 
     
     
         21 . A method for selecting a subject-specific plurality of epitopes for preparing a cancer cell specific pharmaceutical composition to treat cancer in a single subject, the method comprising
 (a) (i) whole genome sequencing or whole exome sequencing cancer cell nucleic acids from a first biological sample comprising cancer cells from the single subject, thereby obtaining a first plurality of nucleic acid sequences comprising cancer cell nucleic acid sequences; and
 (ii) whole genome sequencing or whole exome sequencing non-cancer cell nucleic acids from a second biological sample comprising non-cancer cells from the single subject, thereby obtaining a second plurality of nucleic acid sequences comprising non-cancer cell nucleic acid sequences; 
   (b) identifying a plurality of cancer specific nucleic acid sequences that are specific to the cancer cells of the single subject based on a comparison of the first plurality of nucleic acid sequences obtained in (a)(i), to the second plurality of nucleic acid sequences obtained in (a)(ii),
 wherein the identified plurality of cancer specific nucleic acid sequences encodes two or more different MHC-binding epitope sequences of two or more different proteins that are expressed by the cancer cells, and 
 wherein each of the two or more different MHC-binding epitope sequences of the two or more different proteins comprises a cancer specific amino acid mutation that is not present in the non-cancer cells from the single subject; 
   (c) selecting at least two high affinity MHC-binding epitope sequences based on a calculation of binding affinities of the two or more different MHC-binding epitope sequences identified in (b) to an MHC protein encoded by an HLA allele of the single subject by an MHC peptide binding analysis using a program implemented on a computer system, wherein each of the at least two high affinity MHC-binding epitope sequences is calculated to bind to a protein encoded by an HLA allele of the single subject with an IC 50  of less than 500 nM.   
     
     
         22 . The method of  claim 21 , wherein the method further comprises formulating the pharmaceutical composition, wherein the pharmaceutical composition comprises one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (c), or one or more polynucleotides encoding one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (c). 
     
     
         23 . The method of  claim 22 , wherein formulating comprises formulating the pharmaceutical composition with an adjuvant. 
     
     
         24 . The method of  claim 23 , wherein the adjuvant comprises poly I:poly C. 
     
     
         25 . The method of  claim 22 , wherein each of the at least two high affinity MHC-binding epitope sequences is present in the pharmaceutical composition at an amount of from 50 μg to 1.5 mg. 
     
     
         26 . The method of  claim 21 , wherein a tumor-specific T cell response is induced in the single subject. 
     
     
         27 . The method of  claim 21 , wherein the method further comprises administering an immunostimulatory agent or an anti-immunosuppressive agent. 
     
     
         28 . The method of  claim 27 , wherein the immunostimulatory agent or the anti-immunosuppressive agent is selected from the group consisting of an anti-CTLA-4 agent, an anti-PD1 agent, an anti-PD-L1 agent, an anti-CD25 agent, and an indoleamine (2,3)-dioxygenase (IDO) inhibitor. 
     
     
         29 . The method of  claim 22 , wherein
 (i) each of the one or more polypeptides that comprises a high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) has a length of from 8 to 50 naturally occurring amino acids, or   (ii) each of the one or more polynucleotides that encodes a polypeptide comprising a high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) encodes a polypeptide that has a length of from 8 to 50 naturally occurring amino acids.   
     
     
         30 . The method of  claim 29 , wherein
 (i) each of the one or more polypeptides that comprises a high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) is greater than 15 amino acids in length, or   (ii) each of the one or more polynucleotides that encodes a polypeptide comprising a high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) encodes a polypeptide that is greater than 15 amino acids in length.   
     
     
         31 . The method of  claim 21 , wherein
 (i) a first high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) is predicted to bind to a protein encoded by a first HLA allele of the single subject; and   (ii) the first high affinity MHC-binding epitope sequence or a second high affinity MHC-binding epitope sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) is predicted to bind to a protein encoded by a second HLA allele of the single subject that is different than the first HLA allele.   
     
     
         32 . The method of  claim 22 , wherein formulating comprises
 (a) expressing or synthesizing the one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (c); or   (b) producing the one or more polynucleotides encoding the one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (c).   
     
     
         33 . The method of  claim 21 , wherein the protein encoded by an HLA allele of the single subject is an MHC class I protein, and wherein each of the at least two high affinity MHC-binding epitope sequences selected in (c) has a length of from 8 to 12 naturally occurring amino acids. 
     
     
         34 . The method of  claim 21 , wherein the cancer specific amino acid mutation that is not present in the non-cancer cells from the single subject is encoded by a frameshift mutation. 
     
     
         35 . The method of  claim 21 , wherein each of the at least two high affinity MHC-binding epitope sequences is calculated to bind to a protein encoded by an HLA allele of the single subject with an IC 50  of 100 nM or less. 
     
     
         36 . The method of  claim 21 , wherein the at least two high affinity MHC-binding epitope sequences selected in (c) comprises at most 20 high affinity MHC-binding epitope sequences. 
     
     
         37 . The method of  claim 21 , wherein the at least two high affinity MHC-binding epitope sequences selected in (c) comprises at least 4 high affinity MHC-binding epitope sequences for administration. 
     
     
         38 . The method of  claim 21 , wherein identifying comprises identifying the two or more different proteins that are expressed by cancer cells of the single subject by measuring levels of RNA encoding the two or more different proteins in the cancer cells of the single subject. 
     
     
         39 . The method of  claim 21 , wherein (i) an affinity of one or more of the at least two high affinity MHC-binding epitope sequences selected in (c) to the protein encoded by an HLA allele of the single subject is not experimentally measured, or (ii) an affinity of T cells of the single subject to a complex of a peptide sequence of the at least two high affinity MHC-binding epitope sequences selected in (c) and a protein encoded by an HLA allele of the single subject is not experimentally measured. 
     
     
         40 . The method of  claim 21 , wherein the cancer cells from the single subject are cancer cells of a solid cancer. 
     
     
         41 . The method of claim  1 , wherein each of the at least two high affinity MHC-binding epitope sequences binds to a protein encoded by an HLA allele of the single subject with an IC 50  of less than 150 nM. 
     
     
         42 . The method of claim  1 , wherein the program implemented on a computer system comprises a validated peptide-MHC binding prediction algorithm. 
     
     
         43 . A method for producing pharmaceutical composition comprising a subject-specific plurality of MHC-binding epitope sequences or one or more polynucleotides encoding the subject-specific plurality of MHC-binding epitope sequences, the method comprising:
 (a) identifying a plurality of cancer specific nucleic acid sequences that are specific to cancer cells of a single subject based on a comparison of
 (A) nucleic acid sequences obtained by whole genome sequencing or whole exome sequencing of cancer cells from the single subject to 
 (B) nucleic acid sequences obtained by whole genome sequencing or whole exome sequencing of non-cancer cells from the single subject, 
 wherein the identified plurality of cancer specific nucleic acid sequences encodes two or more different MHC-binding epitope sequences of two or more different proteins that are expressed by the cancer cells, and 
 wherein each of the two or more different MHC-binding epitope sequences of the two or more different proteins comprise a cancer specific amino acid mutation that is not present in the non-cancer cells from the single subject; 
   (b) selecting at least two high affinity MHC-binding epitope sequences based on a calculation of binding affinities of the two or more different MHC-binding epitope sequences identified in (a), to an MHC protein encoded by an HLA allele of the single subject by an MHC peptide binding analysis using a program implemented on a computer system,
 wherein each of the at least two high affinity MHC-binding epitope sequences is calculated to bind to a protein encoded by an HLA allele of the single subject with an IC50 of less than 500 nM; and 
   (c) producing a subject-specific pharmaceutical composition comprising one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (b), or one or more polynucleotides encoding one or more polypeptides comprising the at least two high affinity MHC-binding epitope sequences selected in (b),
 wherein producing the subject-specific pharmaceutical composition comprises: 
 (i) combining the one or more polypeptides with a pharmaceutically acceptable excipient, wherein the one or more polypeptides are synthesized or expressed from a recombinant polynucleotide that encodes the one or more polypeptides, or 
 (ii) combining the one or more polynucleotides with a pharmaceutically acceptable excipient, wherein the one or more polynucleotides are recombinant. 
   
     
     
         44 . A subject-specific pharmaceutical composition that is specific to cancer cells of a single subject with cancer comprising:
 (a) a plurality of selected cancer neoantigen peptides, wherein one or more of the plurality of selected cancer neoantigen peptides induces a cancer-specific T cell response in the single subject, and an adjuvant, or   (b) one or more polynucleotides encoding the plurality of selected cancer neoantigen peptides; wherein
 (i) the plurality of selected cancer neoantigen peptides is from about 13-20 selected cancer neoantigen peptides; 
 (ii) each of the plurality of selected cancer neoantigen peptides is about 15-30 amino acids in length; and 
 (iii) the cancer is a solid cancer; and 
   wherein each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides:
 (A) comprises a neoepitope that is predicted to bind to a protein encoded by an HLA allele expressed by the single subject with an IC50 less than 500 nM, 
 (B) comprises a mutation that is not present in non-cancer cells of the single subject, and 
 (C) is encoded by an expressed gene of the cancer cells of the single subject, 
   wherein the mutation of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides comprises:
 (A) a point mutation, 
 (B) a splice-site mutation, 
 (C) a frameshift mutation, 
 (D) a read-through mutation, or 
 (E) a gene-fusion mutation; 
 and 
   wherein the plurality of selected cancer neoantigen peptides or the one or more polynucleotides encoding the plurality of selected cancer neoantigen peptides are selected by a process comprising:
 (1) selecting a plurality of nucleic acid sequences from a pool of nucleic acid sequences sequenced from the cancer cells of the single subject that encodes a plurality of candidate peptides comprising one or more different mutations not present in a pool of nucleic acid sequences sequenced from non-cancer cells of the single subject, wherein each candidate peptide comprises one or more mutations, and wherein the pool of nucleic acid sequences sequenced from the cancer cells of the single subject and the pool of nucleic acid sequences sequenced from the non-cancer cells of the single subject are sequenced by whole genome sequencing or whole exome sequencing; 
 (2) predicting which candidate peptide sequences of the plurality of candidate peptide sequences comprise a neoepitope that binds to the protein encoded by an HLA allele expressed by the single subject with an IC 50  less than 500 nM by an MHC peptide binding analysis using a program implemented on a computer system; and 
 (3) selecting the plurality of selected cancer neoantigen peptides or the one or more polynucleotides encoding the plurality of selected cancer neoantigen peptides from the candidate peptide sequences based on the MHC peptide binding analysis. 
   
     
     
         45 . The subject-specific pharmaceutical composition of  claim 44 , wherein a first selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is predicted to bind to a protein encoded by a first HLA allele expressed by the single subject and a second selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is predicted to bind to a protein encoded by a second HLA allele expressed by the single subject that is different than the first HLA allele. 
     
     
         46 . The subject-specific pharmaceutical composition of  claim 44 , wherein the pharmaceutical composition further comprises an anti-TNFR superfamily agent. 
     
     
         47 . The subject-specific pharmaceutical composition of  claim 44 , wherein the neoepitope of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is predicted to bind to the protein encoded by an HLA allele of the single subject with an IC 50  less than 250 nM. 
     
     
         48 . The subject-specific pharmaceutical composition of  claim 47 , wherein the neoepitope of each selected neoantigen peptides of the plurality of selected cancer neoantigen peptides is predicted to bind to the protein encoded by an HLA allele of the single subject with an IC 50  less than 100 nM. 
     
     
         49 . The subject-specific pharmaceutical composition of  claim 44 , wherein selecting comprises comparing the plurality of nucleic acid sequences from a first pool of nucleic acid sequences sequenced from the cancer cells of the single subject to a plurality of nucleic acid sequences from a second pool of nucleic acid sequences sequenced from the non-cancer cells of the single subject. 
     
     
         50 . The subject-specific pharmaceutical composition of  claim 44 , wherein the composition further comprises an anti-immunosuppressive agent. 
     
     
         51 . The subject-specific pharmaceutical composition of  claim 50 , wherein the anti-immunosuppressive agent is an anti-CTLA-4 agent, an anti-PD1 agent, an anti-PD-L1 agent, an anti-CD25 agent or an indoleamine (2,3)-dioxygenase (IDO) inhibitor. 
     
     
         52 . The subject-specific pharmaceutical composition of  claim 45 , wherein the first HLA allele expressed by the single subject is a class I HLA allele and the second HLA allele expressed by the single subject is a class I HLA allele or a class II HLA allele. 
     
     
         53 . The subject-specific pharmaceutical composition of  claim 45 , wherein a complex of a neoepitope of a selected neoantigen peptide and the protein encoded by an HLA allele of the single subject is recognized by a T cell receptor of a T-lymphocyte of the single subject. 
     
     
         54 . The subject-specific pharmaceutical composition of  claim 44 , wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, bladder cancer, and melanoma. 
     
     
         55 . The subject-specific pharmaceutical composition of  claim 44 , wherein each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is present in the composition at an amount of from 50 μg to 1.5 mg. 
     
     
         56 . The subject-specific pharmaceutical composition of  claim 44 , wherein
 (i) an ability of the neoepitope of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides to bind to the protein encoded by an HLA allele expressed by the single subject is unknown, or   (ii) an ability of a T cell receptor of a T-lymphocyte of the single subject to bind a complex of a neoepitope of a selected neoantigen peptide and the protein encoded by an HLA allele of the single subject is unknown.   
     
     
         57 . The subject-specific pharmaceutical composition of  claim 44 , wherein each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is a synthetic peptide. 
     
     
         58 . The subject-specific pharmaceutical composition of  claim 44 , wherein the protein encoded by an HLA allele of the single subject is a class I HLA protein or a class II HLA protein. 
     
     
         59 . The subject-specific pharmaceutical composition of  claim 44 , wherein the adjuvant comprises a TLR-based adjuvant, a mineral oil based adjuvant, or a combination thereof. 
     
     
         60 . The subject-specific pharmaceutical composition of  claim 44 , wherein the mutation that is not present in non-cancer cells of the single subject is not essential for development or progression of the cancer cells of the single subject. 
     
     
         61 . The subject-specific pharmaceutical composition of  claim 59 , wherein the adjuvant comprises poly I:poly C. 
     
     
         62 . The subject-specific pharmaceutical composition of  claim 44 , wherein the method used to produce the plurality of selected cancer neoantigen peptides further comprises formulating the plurality of selected cancer neoantigen peptides with the adjuvant. 
     
     
         63 . A kit, comprising:
 (a) a first component comprising the pharmaceutical composition of  claim 44 ; and   (b) a second component comprising an anti-immunosuppressive agent or anti-immunostimulatory agent.   
     
     
         64 . The kit of  claim 65 , wherein the anti-immunosuppressive agent or anti-immunostimulatory agent is selected from the group consisting of an anti-CTLA agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-CD25 agent, an indoleamine (2,3)-dioxygenase inhibitor and combinations thereof. 
     
     
         65 . The subject-specific pharmaceutical composition of  claim 44 , wherein the adjuvant comprises poly I:poly C. 
     
     
         66 . The subject-specific pharmaceutical composition of  claim 44 , wherein the neoepitope of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides that comprises a point mutation is predicted to bind to the protein encoded by an HLA allele expressed by the single subject with an IC 50  less than 150 nM. 
     
     
         67 . The subject-specific pharmaceutical composition of  claim 44 , wherein the one or more polynucleotides encoding the selected plurality of selected cancer neoantigen peptides is DNA or mRNA. 
     
     
         68 . The subject-specific pharmaceutical composition of  claim 44 , wherein the one or more polynucleotides encoding the plurality of selected cancer neoantigen peptides comprises two or more polynucleotides each encoding at least one selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides. 
     
     
         69 . The subject-specific pharmaceutical composition of  claim 44 , wherein the one or more polynucleotides encoding the plurality of selected cancer neoantigen peptides comprises a polynucleotide comprising a first sequence encoding a first selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides operatively linked to a second sequence encoding a second selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides. 
     
     
         70 . The subject-specific pharmaceutical composition of  claim 44 , wherein the plurality of selected cancer neoantigen peptides are expressed peptides. 
     
     
         71 . The subject-specific pharmaceutical composition of  claim 44 , wherein the solid cancer is melanoma. 
     
     
         72 . The subject-specific pharmaceutical composition of  claim 44 , wherein the solid cancer is glioblastoma. 
     
     
         73 . The subject-specific pharmaceutical composition of  claim 44 , wherein the mutation of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is (a) a point mutation and the neoepitope of the selected cancer neoantigen peptide is predicted to bind to the protein encoded by an HLA allele expressed by the single subject with an IC 50  of less than 500 nM, or (b) is encoded by a new open reading frame sequence resulting from a frameshift mutation. 
     
     
         74 . The subject-specific pharmaceutical composition of  claim 44 , wherein the mutation of each selected cancer neoantigen peptide of the plurality of selected cancer neoantigen peptides is (a) a point mutation and the neoepitope of the selected cancer neoantigen peptide is predicted to bind to the protein encoded by an HLA allele expressed by the single subject with an IC 50  of 150 nM or less. 
     
     
         75 . A subject-specific pharmaceutical composition that is specific to cancer cells of a single subject, the pharmaceutical composition comprising:
 (I) a polypeptide or polynucleotide encoding the polypeptide,
 wherein the polypeptide comprises a plurality of amino acid sequences, 
 wherein each amino acid sequence of the plurality of amino acid sequences has formula:
   X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -[X] n    
 wherein each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and [X] n  represents a natural amino acid, 
 wherein n is an integer of from 1 to 43, 
 wherein at least one of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and [X] n  of each amino acid sequence of the plurality of amino acid sequences is a mutant amino acid; and 
 
 wherein at least two of the amino acid sequences of the plurality of amino acid sequences: 
 (a) are encoded by at least two different genes of cancer cells of the single subject, 
 (b) are present in at least two different proteins expressed by the cancer cells of the single subject, 
 (c) are not encoded by the at least two different genes of non-cancer cells of the single subject; and 
   (II) a pharmaceutically acceptable carrier.