Heteroclitic neoepitope vaccines

Abstract

Compositions of peptides modified to improve HLA binding or T cell recognition while conserving reactivity to the target neoepitope, enhance activation of neoantigen-specific T cells. Methods of use include treating cancer by administering one or more of these peptides generated by computer modeling.

Description

Docket No.: 64421-706.601HETEROCLITIC NEOEPITOPE VACCINESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 707,020, entitled “HETEROCLITIC NEOEPITOPE VACCINES”, filed on October 14, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 13, 2025, is named 64421 -706601_SL. xml and is 77,824 bytes in size.BACKGROUND

[0003] Many cancer neoantigens are poorly immunogenic due to weak binding of the neoepitope to human leukocyte antigen (HLA) thereby limiting presentation to cognate T cells. Modifications to the peptide amino acid sequence can improve HLA binding or T cell recognition while conserving reactivity to the target neoepitope, thereby enhancing activation of neoantigen-specific T cells. These modified epitopes, which improve antigen immunogenicity, are termed heteroclitic epitopes or altered peptide ligands (APLs).SUMMARY

[0004] An aspect of the present disclosure is a method of identifying heteroclitic neoepitopes having improved immunogenicity, the method comprising: modeling a library of peptides onto a human leukocyte antigen (HLA) to form an HLA-peptide structure, wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes; (a) selecting the HLA-peptide structure having a sequence identity similar to a parental epitope being modeled; (b) modeling heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position; (c) generating HLA-heteroclitic neoepitope complexes; (d) validating the HLA- heteroclitic neoepitope complexes using a root-mean-square deviation analysis (Ca-RMSD); and (e) performing a high-resolution Monte Carlo simulation with minimization docking of the heteroclitic neoepitope to the HLA cleft to identify the lowest full-atom energy conformation; thereby identifying the heteroclitic neoepitopes having improved immunogenicity. An aspect of the present disclosure is a method of identifying heteroclitic neoepitopes having improvedWSGR Docket No.: 64421-706.601 immunogenicity, the method comprising: (a) modeling a library of peptides onto a human leukocyte antigen (HLA) to form an HLA-peptide structure, wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes; (b) selecting the HLA-peptide structure having a sequence identity similar to a parental epitope being modeled; (c) modeling heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position; (d) generating HLA-heteroclitic neoepitope complexes; (e) validating the HLA-heteroclitic neoepitope complexes using a root-mean-square deviation analysis (Ca-RMSD); and (f) performing a high-resolution Monte Carlo simulation with minimization docking of the heteroclitic neoepitope to the HLA cleft to identify the lowest full-atom energy conformation; thereby identifying the heteroclitic neoepitopes having improved immunogenicity. In some embodiments, the modeling of the peptides onto the HLA comprises aligning peptide sequences with a consensus binding motif of HLA molecules. In some embodiments, modeling the library of peptides onto the HLA comprises aligning peptide sequences with a consensus binding motif of HLA molecules. In some embodiments, the consensus binding motif comprises a consensus binding motif for 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14- mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes. In some embodiments, the consensus binding motif for the 8-mer, 9-mer, 10- mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21- mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprises distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors. In some embodiments, the root-mean-square deviation analysis is conducted to identify unstable structural changes of an HLA cleft. In some embodiments, anchor energetic-optimized structures are selected to identify anchor residue modifications. In some embodiments, the heteroclitic neoepitope complexes are generated to comprise combinations of anchor residue amino acids specific for each HLA class and subgroups thereof. In some embodiments, the HLA comprises an HLA type, wherein the HLA type comprises class I, II, or a subgroup thereof. In some embodiments, the peptides are derived from tumor cells or antigenic derivatives thereof. In some embodiments, the peptides comprise one or more tumor antigens. In some embodiments, the one or more tumor antigens comprise one or more of tumor protein 53 (TP53), isocitrate dehydrogenase 1 (IDH1), catenin beta 1 (CTNNB1), epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2), Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma oncogene homolog Bl (BRAF), F-box and WD repeat domain containing 7 (FBWX7), fibroblast growth factor receptor 3 (FGFR3), mucin 4 (MUC4),WSGR Docket No.: 64421-706.601 neuroblastoma RAS viral oncogene homolog (NRAS), phosphatidylinositol-4,5-bisphosphate 3 -kinase catalytic subunit alpha (PIK3CA), protein phosphatase 2 regulatory subunit A alpha (PPP2R1A), or phosphatase and tensin homolog (PTEN). In some embodiments, the one or more tumor antigens comprise one or more mutations. In some embodiments, the one or more mutations comprise TP53 Y220C. In some embodiments, the one or more mutations comprise IDH1 R132H. In some embodiments, the one or more mutations comprise CTNNB1 S45F. In some embodiments, the one or more mutations comprise EGFR L858R. In some embodiments, the one or more mutations comprise JAK2 V617F. In some embodiments, the HLA is HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA-A* 11 :01, HLA-A*30:01, or HLA-A*68:01. In some embodiments, the HLA-B is HLA- B*07:02. In some embodiments, the HLA-C is HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, or HLA-C*08:02. In some embodiments, the HLA comprises HLA-A* 11 :01, HLA-A*01 :01, HLA-A*02:01, HLA-A*03:01, HLA-A*26:01, HLA-A*29:02, HLA-A*32:01, HLA- A*24:02, HLA-A*33:03, HLA-A*68:01, HLA-A*31 :01 or HLA-A*02:06. In some embodiments, the HLA comprises HLA-B*13:02, HLA-B*41 :01, HLA-B*18:03, HLA- B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51:01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01 or HLA-B*52:01. In some embodiments, the HLA comprises HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01 :02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C*12:03 or HLA-C* 14:02. In some embodiments, the HLA comprises HLA-A*01 :01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31 :01, HLA-A*32:01, HLA- A*33:01, HLA-A*68:01, HLA-A*l l :01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA- B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B* 13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41 :02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01 :02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C* 16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA- C*17:01. In some embodiments, the HLA-peptide structure shares a sequence identity of at least 85%, 90%, 95%, 97%, or 99% with the parental epitope.WSGR Docket No.: 64421-706.601

[0005] An aspect of the present disclosure is a method of generating a library of immunogenic peptides, comprising: modeling a peptide onto a human leukocyte antigen (HLA), wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes; selecting an HLA-peptide structure having a sequence identity similar to a parental epitope being modeled; modeling a heteroclitic neoepitope into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position to form HLA-heteroclitic neoepitope complexes; validating the resulting HLA-heteroclitic neoepitope complexes using a root-mean-square deviation analysis (Ca-RMSD); and performing a high-resolution Monte Carlo simulation with minimization docking of the heteroclitic neoepitope to the HLA cleft to identify the lowest full-atom energy conformation; thereby generating the library of immunogenic peptides. In some embodiments, the modeling comprises introducing amino acid rotamers with a lowest energy conformation at each position. In some embodiments, the root-mean-square deviation analysis is conducted to identify unstable structural changes of the HLA cleft. In some embodiments, the peptide epitopes comprise peptides isolated from tumor cells or antigenic derivatives thereof. In some embodiments, the peptide epitopes comprise one or more tumor antigens. In some embodiments, the one or more tumor antigens comprise at least a portion of an amino acid sequence of one or more of TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN. In some embodiments, the one or more tumor antigens comprises one or more mutations. In some embodiments, the one or more mutations comprise TP53 Y220C. In some embodiments, the one or more mutations comprise IDH1 R132H. In some embodiments, the one or more mutations comprise CTNNB1 S45F. In some embodiments, the one or more mutations comprise EGFR L858R. In some embodiments, the one or more mutations comprise JAK2 V617F. In some embodiments, a sequence of a tumor antigen selected from the one or more tumor antigens is positionally aligned with 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20- mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes that bind HLA molecules. In some embodiments, the 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprise distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors. In some embodiments, a tumor antigen peptide comprising the greatest sequence similarity is selected for modeling into an HLA cleft. In some embodiment, the heteroclitic neoepitope comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,WSGR Docket No.: 64421-706.601 at least 22, at least 23, at least 24, at least 25, or more amino acid residues. In some embodiment, the heteroclitic neoepitope comprises at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25 amino acid residues. In some embodiments, anchor energetic-optimized HLA-heteroclitic neoepitope structures are selected to identify anchor residue modifications. In some embodiments, a library of tumor antigen peptides is generated comprising combinations of anchor residue amino acids specific for each HLA class and subgroups thereof. In some embodiments, the tumor antigen peptides comprise heteroclitic epitopes. In some embodiments, contacting the tumor antigen peptides comprising heteroclitic epitopes with a population of immune cells leads to a higher percentage of expansion of the population of immune cells as compared to contacting with corresponding peptides that are otherwise the same but do not comprise the heteroclitic epitopes. In some embodiments, contacting the tumor antigen peptides comprising heteroclitic epitopes with a population of immune cells leads to higher activation of the population of immune cells as compared to contacting with corresponding peptides that are otherwise the same but do not comprise the heteroclitic epitopes. In some embodiments, the activation comprises IFNg secretion. In some embodiments, the HLA comprises an HLA type, wherein the HLA type comprises class I, class II, or a subgroup thereof. In some embodiments, the HLA is HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA-A* 11 :01, HLA-A*30:01, or HLA-A*68:01. In some embodiments, the HLA-B is HLA- B*07:02. In some embodiments, the HLA-C is HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, or HLA-C*08:02. In some embodiments, the HLA comprises HLA-A* 11 :01, HLA-A*01 :01, HLA-A*02:01, HLA-A*03:01, HLA-A*26:01, HLA-A*29:02, HLA-A*32:01, HLA- A*24:02, HLA-A*33:03, HLA-A*68:01, HLA-A*31 :01 or HLA-A*02:06. In some embodiments, the HLA comprises HLA-B*13:02, HLA-B*41 :01, HLA-B*18:03, HLA- B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51 :01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01 or HLA-B*52:01. In some embodiments, the HLA comprises HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01 :02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C*12:03 or HLA-C* 14:02. In some embodiments, the HLA comprises HLA-A*01 :01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31 :01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*l l :01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-WSGR Docket No.: 64421-706.601B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B* 13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01 :02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C* 16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA- C* 17:01. In some embodiments, the method further comprises quantifying differences in structural features between the peptide being modeled and the parental epitope. In some embodiments, the structural features comprise contacts between the peptide and HLA cleft residues, solvent-accessible surface area (SASA), peptide rigidity as a measure of CaRMSD of the top 10 predicted structural models for each HLA-heteroclitic neoepitope complex, surface hydrophobicity, or electrostatic potential. In some embodiments, the method further comprises in vitro co-culturing of heteroclitic peptides with HLA-matched lymphocytes to validate which heteroclitic peptides induce the greatest T cell expansion and activation as compared to parental peptides, as assessed by IFN-gamma ELISpot assay.

[0006] An aspect of the present disclosure is a method of treating cancer comprising administering to a subject in need thereof one or more peptides comprising one or more heteroclitic epitopes. In some embodiments, the one or more peptides are generated by the any one of the methods disclosed herein. In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the method further comprises administering one or more therapeutics to the subject.

[0007] An aspect of the present disclosure is a cancer vaccine comprising one or more peptides, wherein the one or more peptides comprise at least one heteroclitic neoepitope. In some embodiments, the one or more peptides comprise a tumor antigen. In some embodiments, the tumor antigen comprises TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1A, or PTEN. In some embodiments, the tumor antigen comprises one or more mutations. In some embodiments, the one or more mutations comprise TP53 Y220C. In some embodiments, the one or more mutations comprise IDH1 R132H. In some embodiments, the one or more mutations comprise CTNNB1 S45F. In some embodiments, the one or more mutations comprise EGFR L858R. In some embodiments, the one or more mutations comprise JAK2 V617F. In some embodiments, the one or more peptides are displayed on a surface of an antigen presenting cell (APC) via an HLA antigen presentationWSGR Docket No.: 64421-706.601 pathway, and wherein a tumor specific immune response is induced. In some embodiments, the one or more peptides are immunogenic specifically for the subject’s HLA type. In some embodiments, the HLA is HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA-A* 11:01, HLA-A*30:01, or HLA-A*68:01. In some embodiments, the HLA-B is HLA-B*07:02. In some embodiments, the HLA-C is HLA- C*01:02, HLA-C*03:03, HLA-C*03:04, or HLA-C*08:02. In some embodiments, the HLA comprises HLA-A* 11:01, HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*26:01, HLA-A*29:02, HLA-A*32:01, HLA-A*24:02, HLA-A*33:03, HLA-A*68:01, HLA-A*31:01 or HLA-A* 02: 06. In some embodiments, the HLA comprises HLA-B*13:02, HLA-B*41:01, HLA-B*18:03, HLA-B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51:01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01 or HLA-B*52:01. In some embodiments, the HLA comprises HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01 :02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C* 12:03 or HLA-C* 14:02. In some embodiments, the HLA comprises HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA- A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*l l:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA- B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C* 16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:01. In some embodiments, the at least one heteroclitic neoepitope is generated by any one of the methods disclosed herein. In some embodiments, the one or more peptides comprise one or more immunogenic peptides generated by any one of the methods disclosed herein.

[0008] An aspect of the present disclosure is an expression vector encoding the one or more peptides disclosed herein.

[0009] An aspect of the present disclosure is a method of treating cancer in a subject diagnosed with the cancer comprising: isolating cells from a biological sample of the subject;WSGR Docket No.: 64421-706.601 culturing the isolated cells with one or more peptide generated by any one of the methods disclosed herein, isolating T cells, NK cells, and / or antigen presenting cells cultured with the one or more peptide and expanding the T cells, NK cells, and / or antigen presenting cells to produce a therapeutically effective composition of tumor antigen specific T cells, NK cells, and / or antigen presenting cells; adoptively transferring the tumor antigen specific T cells, NK cells, and / or antigen presenting cells into the subject, thereby treating the subject diagnosed with cancer. In some embodiments, the one or more peptides comprise one or more heteroclitic neoepitopes.

[0010] An aspect of the present disclosure is a cell comprising one or more peptides generated by any one of the methods disclosed herein.

[0011] An aspect of the present disclosure is a pharmaceutical composition comprising one or more of a peptide generated by any one of the methods disclosed herein, a cancer vaccine as disclosed herein, a cell as disclosed herein, an expression vector as disclosed herein, and a pharmaceutically acceptable excipient.

[0012] An aspect of the present disclosure is the use of one or more peptides generated by any one of the methods disclosed herein, a cancer vaccine as disclosed herein, an expression vector as disclosed herein, a cell as disclosed herein, and / or a pharmaceutical composition as disclosed herein in the manufacture of a medicament for treatment in a subject. In some embodiments, any one of the methods disclosed herein further comprise quantifying differences in structural features between the peptide being modeled and the parental epitope. In some embodiments, the features comprise contacts between the peptide and HLA cleft residues, solvent-accessible surface area (SASA), peptide rigidity as a measure of CaRMSD of the top 10 predicted structural models for each HLA-heteroclitic neoepitope peptide complex, surface hydrophobicity, or electrostatic potential. In some embodiments, any one of the methods disclosed herein further comprise in vitro co-culturing of heteroclitic peptides with HLA-matched lymphocytes to validate which heteroclitic peptides induce the greatest T cell expansion and activation as compared to parental peptides, as assessed by IFN-gamma ELISpot assay.

[0013] An aspect of the present disclosure is a kit comprising: (a) one or more peptides identified by any one of the methods disclosed herein, a cancer vaccine as disclosed herein, an expression vector as disclosed herein, a cell as disclosed herein, a pharmaceutical composition as disclosed herein, and (b) an information material containing instructions for administering a dosage of the one or more peptides, the cancer vaccine, the cell, or a dosage form of the pharmaceutical composition to a subject.WSGR Docket No.: 64421-706.601

[0014] An aspect of the present disclosure is a T cell receptor obtained via immunizing a subject with any one of the peptides of the present disclosure and characterizing a subsequent immune response.

[0015] In some embodiments of any of the above aspects, the cancer is selected from the group consisting of: colorectal cancer, leukemia, Kaposi’s sarcoma, breast cancer, bone cancers, brain cancer, mantle cell lymphoma, non-Hodgkin’s lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, retinoblastoma, esophagus cancer, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, sarcomas, Wilms’ tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, and multidrug resistant cancer. In some embodiments, the leukemia is selected from the group consisting of: acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia. In some embodiments, the bone cancer is selected from the group consisting of: osteosarcoma, chondrosarcomas, Ewing’s sarcoma, fibrosarcomas, giant cell tumors, adamantinomas, and chordomas. In some embodiments, the brain cancer is selected from the group consisting of: meningiomas, glioblastomas, lower-grade astrocytomas, oligodendrocytomas, pituitary tumor, schwannomas, primary CNS lymphoma, and metastatic brain cancers. In some embodiments, the lung cancer is non-small cell lung carcinoma.

[0016] Definitions

[0017] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. Definitions of common terms can be found in Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons New York, NY (2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons New York, NY (2001); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012); Jon Lorsch (ed.) Laboratory Methods inWSGR Docket No.: 64421-706.601Enzymology: DNA, Elsevier, (2013); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, (2014); John E. Coligan (ed.), Current Protocols in Protein Science (CPPS), John Wiley and Sons, Inc., (2005); and Ethan M Shevach, Warren Strobe, (eds.) Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, John Wiley and Sons, Inc., (2003); each of which provide one skilled in the art with a general guide to many of the terms used in the present application.

[0018] Standard nomenclature is used for the natural amino acids and their abbreviations. For example, L-alanine is represented with the three-letter abbreviation Ala, or one-letter abbreviation “A”. Where indicated, the “D” stereoisomer of alanine is represented as D-Ala.

[0019] Standard nomenclature is used for the bases of DNA, with cytosine, guanosine, adenine, and thymine indicated as “C”, “G”, “A”, and “T”, and codons that encode DNA follow the standard genetic code, for example the amino acid Leu is encoded by TTA, TTG, CTT, CTC, CTA or CTG, and Asp is encoded by GAT or GAC.

[0020] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0021] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and also within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. All numeric values are herein assumed to be modified by the term “about”, whether or not explicitly indicated. The recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0022] As used herein, an “adjuvant” refers to a substance that enhances the body's immune response to an antigen or a vaccine and may be added to the formulation that includes theWSGR Docket No.: 64421-706.601 immunizing agent. Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine. Adjuvants may include, for example, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabolizable oil, mineral and / or plant / vegetable and / or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid saccharide crosslinked polymer), HRA-3 with cottonseed oil (CSO), or an acrylic acid polyol cross-linked polymer. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate / caprate), glyceryl tri -(capryl ate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers comprise nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the PLURONIC™ brand products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.) John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). In a preferred embodiment the adjuvant is at a concentration of about 0.01 to about 50%, at a concentration of about 2% to 30%, at a concentration of about 5% to about 25%, at a concentration of about 7% to about 22%, and at a concentration of about 10% to about 20% by volume of the final product. “Adjuvanted” refers to a composition that incorporates or is combined with an adjuvant.

[0023] In the description and in the claims, phrases such as “at least one of’ or “one or more of’ may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including threeWSGR Docket No.: 64421-706.601 or more items. For example, the phrases “at least one of A, B, and C”, “one or more of A, B, and C” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0024] The term “HLA-peptide structure” as used herein refers to a complex where a peptide is bound to one or a plurality of HLA-haplotypes. The term “amino acid” as used herein refers to naturally occurring and synthetic a, P, y, and 5 amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenyl al aninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, P-alanyl, P-valinyl, P-leucinyl, P-isoleucinyl, P-prolinyl, P-phenylalaninyl, P-tryptophanyl, P-methioninyl, P-glycinyl, P-serinyl, P- threoninyl, P-cysteinyl, P-tyrosinyl, P-asparaginyl, P-glutaminyl, P-aspartoyl, P-glutaroyl, P- lysinyl, P-argininyl or P-histidinyl. The amino acids can be non-naturally occurring amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, D- amino acids (i.e. an amino acid of an opposite chirality to the naturally-occurring form), N-oc- methyl amino acids, C-a-methyl amino acids, P-methyl amino acids and D- or L-P-amino acids. Other non-naturally occurring amino acids include, for example, P-alanine (P-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (y-Abu), 2- aminoisobutyric acid (Aib), 6-aminohexanoic acid (s-Ahx), ornithine (om), sarcosine, a-amino isobutyric acid, 3 -aminopropionic acid, 2,3 -diaminopropionic acid (2,3-diaP), D- or L- phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of a, P, y, and 8 glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.

[0025] By “cancer” as used herein is meant, a disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including colorectal cancer, as well as, for example, leukemias, e.g., acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andWSGR Docket No.: 64421-706.601 chronic lymphocytic leukemia, AIDS related cancers such as Kaposi’s sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing’s sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, Primary CNS Lymphoma, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms’ tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, and other cancer or proliferative disease, condition, trait, genotype or phenotype.

[0026] The term “combination therapy”, as used herein, refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents. When used in combination therapy, two or more different agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations. In another alternative, a first agent can be administered just followed by one or more additional agents. In the separate administration protocol, two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart. Treatment of a subject also includes a variety of combination therapies with both physical, e.g. surgery, and radiation based treatments.WSGR Docket No.: 64421-706.601

[0027] As used herein, the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.” By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.

[0028] “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0029] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0030] A “heteroclitic epitope” or “heteroclitic analog” or "heteroclitic neoepitope” or “altered peptide ligand (APL)” are used interchangeably herein, and refers to an altered version of an endogenous peptide sequence (i.e., an analog) engineered to elicit potent immune reactions. Heteroclitic epitopes have increased stimulatory capacity or potency for a specific T cell, as measured by increased responses to a given dose, or by a requirement of lesser amounts to achieve the same response and therefore provide benefit as vaccine components since these epitopes induce T cell responses stronger than those induced by the native epitope. In some embodiments, the potent immune reactions improved HLA binding. In some embodiments, the potent immune reactions comprises improved T cell recognition. In some embodiments, the heteroclitic neoepitopes are derived from tumor antigens or cancer neoantigen.WSGR Docket No.: 64421-706.601

[0031] An “immunogenic peptide” or “antigenic peptide” are used interchangeably herein, and refers to a peptide or epitope that can be recognized by the immune system and elicit an immune response. Immunogenic peptides or antigenic peptides may comprise a motif such that the peptide will bind an MHC molecule and induce a T cell response, or can be recognized by the B cell receptor on the B cell to induce antibody production.

[0032] An “immunogenic epitope” or “antigenic epitope” are used interchangeably herein, and refers to a part of an antigen is recognized by the immune system, e.g., by antibodies, B cells, or T cells. In some embodiments, the epitope is the specific piece of the antigen to which an antibody binds. Although epitopes are usually non-self proteins, sequences derived from the host can, in some instances, be recognized.

[0033] As used herein, the term “immune cells” refers to any cells of the immune system that are involved in mediating an immune response. Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof. In some aspects, an immune cell expresses CD3. In certain aspects, the CD3 -expressing immune cells are T cells (e.g., CD4+T cells or CD8+T cells). In some aspects, an immune cell that can be targeted with a targeting moiety (e.g., anti-CD3) comprises a naive CD4+T cell. In some aspects, an immune cell comprises a memory CD4+T cell. In some aspects, an immune cell comprises an effector CD4+T cell. In some aspects, an immune cell comprises a naive CD8+T cell. In some aspects, an immune cell comprises a memory CD8+T cell. In some aspects, an immune cell comprises an effector CD8+T cell. In some aspects, an immune cell comprises a gamma delta T cell. In some aspects, an immune cell is a dendritic cell. In certain aspects, a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDCl), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.

[0034] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell and are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.WSGR Docket No.: 64421-706.601

[0035] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intravitreal (i.v.i.), intra-cistema magna (i.c.m.), or intrastemal injection, or infusion techniques.

[0036] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0037] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In embodiments, the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0038] By “proteins or molecules of the major histocompatibility complex (MHC)”, “MHC molecules”, “MHC proteins” or “HLA proteins” is thus meant proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells. In some embodiments, MHC molecules of class I consist of a heavy chain and a light chain and are capable of binding a peptide of about 8 to 11 amino acids, but usually 9 or 10 amino acids, if this peptide has suitable binding motifs, and presenting it to cytotoxic T-lymphocytes. In some embodiments, the peptide bound by the MHC molecules of class I originates from an endogenous protein antigen. In some embodiments, the heavy chain of the MHC molecules of class I is preferably an HLA- A, HLA-B or HLA-C monomer, and the light chain is P-2-microglobulin (P2M).

[0039] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.WSGR Docket No.: 64421-706.601

[0040] The terms “sample,” “patient sample,” “biological sample,” and the like, encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic, prognostic and / or monitoring assay. The patient sample may be obtained from a healthy subject, a diseased patient, or a patient with lung cancer. In certain embodiments, a sample that is “provided” can be obtained by the person (or machine) conducting the assay, or it can have been obtained by another, and transferred to the person (or machine) carrying out the assay. Moreover, a sample obtained from a patient can be divided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis. The definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, cord blood, amniotic fluid, cerebrospinal fluid, urine, saliva, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. In certain embodiment, a sample comprises cerebrospinal fluid. In a specific embodiment, a sample comprises a blood sample. In another embodiment, a sample comprises a plasma sample. In yet another embodiment, a serum sample is used. The definition of “sample” also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations. The terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and / or formalin-fixed, paraffin- embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.

[0041] The terms “treat,” “treated,” “treating,” “treatment,” and the like are meant to refer to reducing or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor). “Treating” may refer to administration of the therapy to a subject after the onset, or suspected onset, of a cancer. “Treating” includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a cancer and / or the side effects associated with cancer therapy. The term “treating” also encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.WSGR Docket No.: 64421-706.601

[0042] The term “therapeutic effect” refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology. “Therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0043] The term “transfected” or “transformed” or “transduced” means to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The transfected / transformed / transduced cell includes the primary subject cell and its progeny.

[0044] As used herein, an “unnatural amino acid,” “non-natural amino acid”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids.

[0045] Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3 -methylphenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p- amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosineWSGR Docket No.: 64421-706.601 amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotinanalogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid containing amino acid; an a, a disubstituted amino acid; a P-amino acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and / or one or more natural amino acids.

[0046] As used herein, “variant” of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

[0047] As used herein, the term “virus” includes any type of virus or virus vector. For example, adenovirus, adeno-associated virus (AAV), recombinant adeno-associated virus (rAAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus,WSGR Docket No.: 64421-706.601 vesicular stomatitis virus, picomavirus. In various embodiments the virus is a chimeric virus, a synthetic virus, a recombinant virus, a mosaic virus or a pseudotyped virus. In some embodiments an AAV / rAAV disclosed herein can AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and / or AAV10 capsid.

[0048] The vaccine of the present disclosure may ameliorate a disease as described herein. By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a neoplasia, tumor, autoimmunity, infection, etc.).

[0049] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants.

[0050] The practice of the present disclosure employs, unless otherwise indicated, techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology. See, e.g., Maniatis etal., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, AWSGR Docket No.: 64421-706.601Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ, of Oregon Press, Eugene, 2000).

[0051] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0053] FIG. 1 is a series of Word plots of 9-mer peptide binding consensus sequences from each HLA allele. Data was accessed through HLAthena browser.

[0054] FIG. 2 shows a heatmap of HLA allele frequency in the United States population.

[0055] FIG. 3A- FIG. 3F show a series of plots and schematics demonstrating that engineered peptides display enhanced contact points and solvent accessible surface area, and reduced rigidity in a murine PDAC model. FIG. 3 A shows that mice were vaccinated twice 7 days apart with PBS, 50 pg APL, or 5 pg parental peptide. Seven days after the last vaccine dose, splenocytes were restimulated with APL or parental peptides, overnight, and IFNy production was measured by ELISpot. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed. FIG. 3B shows in silica structural modeling of APL or parental peptide in murine H2-Kb (murine MHC class I molecule H2-Kb, minimal epitope of peptide 44, parental or engineered amino acid residue). Structural features were analyzed from parental or APL MHC models. Number of contacts between MHC binding cleft and peptide (FIG. 3C) or mutated residue (FIG. 3D) are shown. FIG. 3E shows Ca-RMSD measure ofWSGR Docket No.: 64421-706.601 peptide rigidity. FIG. 3F shows solvent accessible surface area (SASA). Unpaired students t test was performed, (ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0056] FIG. 4A-FIG. 4B demonstrate that KRAS G12D APL1-HLA* 11:01 binding and immunogenicity of engineered antigens show superior binding and T Cell Responses. FIG. 4A shows non-linear regression curves and calculated ECso values for binding between HLA- A* 11 :01 and a parental KRAS G12D 10-mer peptide (SEQ ID NO: 11) and APL (SEQ ID NO: 12). FIG. 4B shows the results of an IFN-y ELISpot assay detecting the induction of KRASmut-specific T cells when treated with no peptide, irrelevant peptide, parental peptide or APL.

[0057] FIG. 5A-FIG. 5B demonstrate that KRAS G12D APL1-HLA* 11:01 binding and immunogenicity of engineered antigens show superior binding and T Cell responses. FIG. 5A shows non-linear regression curves and calculated ECso values for binding between HLA- A* 11 :01 and a parental KRAS G12D 10-mer peptide (SEQ ID NO: 11) and APL (SEQ ID NO: 77). FIG. 5B illustrate the results of an IFN-y ELISpot assay detecting the induction of KRASmut-specific T cells when treated with no peptide, irrelevant peptide, parental peptide or APL.

[0058] FIG. 6 show a series of graphs showing the results obtained from a KRAS APL HLA- A*l l :01 binding assay. Percent binding of KRAS G12D, G12R, G12V, and G12C 10- and 9- mer parental epitopes and altered peptide ligands. Percent binding of each APL is calculated relative to the respective parental epitope.

[0059] FIG. 7 demonstrates the method of identifying heteroclitic neoepitopes described herein.DETAILED DESCRIPTION

[0060] This disclosure relates to the utilization of structural features of neoantigen epitope binding to diverse HLA subtypes to predict optimal anchor modifications that will improve HLA binding and epitope immunogenicity in an HLA-specific manor. The result of this prediction is the optimized heteroclitic peptide vaccine candidate for a patient’s specific HLA type. This technology is applicable for shared neoantigens such as mutant TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q,WSGR Docket No.: 64421-706.601TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y which has been shown to be weakly immunogenic in patients in vaccination settings. In certain aspects, the disclosure provides herein TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y heteroclitic epitope vaccine candidates designed and modelled for diverse HLA subtypes. Additionally, the software described herein is applicable to private patient neoantigen targets where the algorithm can prioritize immunogenic neoantigen targets based on structural features of neoantigen display as well as identify optimal heteroclitic peptides for the patient’s HLA subtypes. In some embodiments, a heteroclitic peptide disclosed herein is selected based on the assessment of one or more of contacts between the peptide and HLA cleft residues, solvent- assessable surface area (SAS A), peptide rigidity as a measure of CaRMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential and / or induction of the greatest T cell expansion and activation as compared to parental peptides. An exemplary workflow for the method is depicted in FIG. 7.

[0061] Immune System and Antigen Presentation

[0062] The immune system can be classified into two functional subsystems: the innate and the acquired immune system. The innate immune system is the first line of defense against infections, and most potential pathogens are rapidly neutralized by this system before they can cause, for example, a noticeable infection. The acquired immune system reacts to molecular structures, referred to as antigens, of the intruding organism. There are two types of acquired immune reactions, which include the humoral immune reaction and the cell-mediated immune reaction. In the humoral immune reaction, antibodies secreted by B cells into bodily fluids bind to pathogen-derived antigens, leading to the elimination of the pathogen through a variety of mechanisms, e.g. complement-mediated lysis. In the cell-mediated immune reaction, T-cells capable of destroying other cells are activated. For example, if proteins associated with a disease are present in a cell, they are fragmented proteolytically to peptides within the cell. Specific cell proteins then attach themselves to the antigen or peptide formed in this manner and transport them to the surface of the cell, where they are presented to the molecular defenseWSGR Docket No.: 64421-706.601 mechanisms, in particular T-cells, of the body. Cytotoxic T cells recognize these antigens and kill the cells that harbor the antigens.

[0063] The molecules that transport and present peptides on the cell surface are referred to as proteins of the major histocompatibility complex (MHC). MHC proteins are classified into two types, referred to as MHC class I and MHC class II. The structures of the proteins of the two MHC classes are very similar; however, they have very different functions. Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells. MHC class I proteins are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells and are then presented to naive or cytotoxic T- lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B -lymphocytes, macrophages and other antigen-presenting cells. They mainly present peptides, which are processed from external antigen sources, i.e. outside of the cells, to T-helper (Th) cells. Most of the peptides bound by the MHC class I proteins originate from cytoplasmic proteins produced in the healthy host cells of an organism itself, and do not normally stimulate an immune reaction. Accordingly, cytotoxic T-lymphocytes that recognize such self-peptide- presenting MHC molecules of class I are deleted in the thymus (central tolerance) or, after their release from the thymus, are deleted or inactivated, i.e. tolerized (peripheral tolerance). MHC molecules are capable of stimulating an immune reaction when they present peptides to non- tolerized T-lymphocytes. Cytotoxic T-lymphocytes have both T-cell receptors (TCR) and CD8 molecules on their surface. T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules of MHC class I. Each cytotoxic T-lymphocyte expresses a unique T-cell receptor which is capable of binding specific MHC / peptide complexes.

[0064] The peptide antigens attach themselves to the molecules of MHC class I by competitive affinity binding within the endoplasmic reticulum, before they are presented on the cell surface. Here, the affinity of an individual peptide antigen is directly linked to its amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence. If the sequence of such a peptide is known, it is possible to manipulate the immune system against diseased cells using, for example, peptide vaccines. The human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. As used herein, “HLA alleles” and “HLA haplotypes” can refer to the MHC proteins encoded by an individual’s HLA system and gene complex.

[0065] The present disclosure provides methods for predicting peptides capable of binding to HLA alleles. The embodiments comprise a set of candidate peptide sequences andWSGR Docket No.: 64421-706.601 identifying one or more structural features indicative of occupancy of the candidate peptides on the binding pocket or cleft of HLA alleles. This is done, for example, by identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes. The HLA-peptide structure having a sequence identity greater than a control HLA- peptide structure is selected and the machine learning algorithm models the heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position. These structural features are input into a machine learning algorithm model to simulate occupancy of one or more binding peptides and one or more nonbinding peptides on the HLA binding pocket in a crystal structure of the HLA allele or the crystal structure of a similar HLA allele. The structural features can be extracted from the output models generated during the simulations. The structural features can also be identified using a machine learning algorithm model that infers occupancy of the one or more candidate peptides on the HLA binding pocket. The inference can be based on the model being trained with simulated models of peptides verified to bind to the HLA allele and verified peptides that do not bind to the HLA allele.

[0066] The present disclosure provides methods for predicting peptides capable of binding to HLA variants encoded by HLA alleles. The embodiments can comprise a set of candidate peptide sequences and identifying one or more structural features indicative of occupancy of the candidate peptides on the binding pocket or cleft of HLA variants. In some embodiments, this can be done, for example, by identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes. The HLA-peptide structure having a sequence identity greater than a control HLA-peptide structure can be selected and the machine learning algorithm models the heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position. These structural features can be input into a machine learning algorithm model to simulate occupancy of one or more binding peptides and one or more non-binding peptides on the HLA binding pocket in a crystal structure of the HLA variant or the crystal structure of a similar HLA variant. The structural features can be extracted from the output models generated during the simulations. The structural features can also be identified using a machine learning algorithm model that infers occupancy of the one or more candidate peptides on the HLA binding pocket. The inference can be based on the model being trained with simulated models of peptides verified to bind to the HLA variant and verified peptides that do not bind to the HLA variant.WSGR Docket No.: 64421-706.601

[0067] Vaccine therapies (e.g., cancer and infections) rely on accurate selection of immunizing peptides to potentiate immune responses (e.g., against tumor-specific neoepitopes or viral epitopes). Given the patient's particular complement of HLA alleles, the ability to predict which epitopes will be presented is a fundamental prerequisite for successful vaccine design. Additionally, given the unique accumulation of mutations in different tumors as well as the patient's particular complement of HLA alleles, the ability to predict which epitopes will be presented is a fundamental prerequisite for successful cancer vaccine design.

[0068] In certain embodiments, an initial input of a candidate peptide or a set of candidate peptides, e g. TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1A, or PTEN peptides is provided. In certain embodiments, the candidate peptide or set of candidate peptides may be obtained from a subject or group of subjects in need of an immune response or modified immune response. In certain embodiments, candidate peptides can be identified in a peptide sequence database (e.g., derived from sequencing of subjects having a specific condition where an immunogenic composition would be useful). In some embodiment, administration of the heteroclitic peptide induces a T-cell- mediated immune response. In some embodiment, administration of the heteroclitic peptide induces expansion of T cells. In some embodiment, administration of the heteroclitic peptide induces activation of T cells.

[0069] In certain embodiments, a peptide sequence database includes HLA allele binding and non-binding peptides. Candidate peptides can be isolated and sequenced for each HLA-allele to identify HLA-binding peptides. In particular embodiments, candidate peptides can be obtained by providing a) a population of cells which expresses a single class I HLA allele or a single pair of class II HLA alleles (one a-chain and one P-chain); b) isolating the respective HLA-peptide complexes from said cells; c) isolating peptides from said HLA-peptide complexes; and d) sequencing the peptides.

[0070] The population of cells may express either a single class I HLA allele, a single pair of class II HLA alleles, or a single class I HLA allele and a single pair of class II HLA alleles. Suitable cell populations include, e.g., class I deficient cells lines in which a single HLA class I allele is expressed, class II deficient cell lines in which a single pair of HLA class II alleles are expressed, or class I and class II deficient cell lines in which a single HLA class I and / or single pair of class II alleles are expressed.WSGR Docket No.: 64421-706.601

[0071] The population of cells may be professional antigen presenting cells such as macrophages, B cells and dendritic cells. In certain embodiments, the cells are B cells or dendritic cells. In certain embodiments, the cells are tumor cells or cells from a tumor cell line. In particular embodiments, the cells are cells isolated from a patient.

[0072] In some embodiments, the population of cells are further modified, such as by increasing or decreasing the expression and / or activity of at least one gene. In certain embodiments, the gene encodes a member of the immunoproteasome. In some embodiments, the immunoproteasome is involved in the processing of HLA class I binding peptides and includes the LMP2 (|3ii), MECL-1 (P2i), and LMP7 (P5i) subunits. In some embodiments, the immunoproteasome can also be induced by interferon-gamma. Accordingly, in some embodiments, the population of cells may be contacted with one or more cytokines, growth factors, or other proteins. In some embodiments, the cells are stimulated with inflammatory cytokines such as interferon-gamma, IL-ip, IL-6, and / or TNF-a. The population of cells may also be subjected to various environmental conditions, such as stress (heat stress, oxygen deprivation, glucose starvation, DNA damaging agents, etc.). In some embodiments, the cells are contacted with one or more of a chemotherapy drug, radiation, targeted therapies or immunotherapy. The methods disclosed herein can therefore be used to study the effect of various genes or conditions on HLA peptide processing and presentation. In particular embodiments, the conditions used are selected so as to match the condition of the patient for which the population of HLA-peptides is to be identified.

[0073] In some embodiments, any HLA allele may be expressed in the cell population. In some embodiments, it will be of interest to sequentially perform the methods provided herein for different HLA alleles, such that resulting datasets can be used in combination. In certain embodiments, the HLA allele is selected so as to correspond to a genotype of interest. In a certain embodiment, the HLA allele is a mutated HLA allele, which may be non-naturally occurring allele or a naturally occurring allele in an afflicted patient. In some embodiments, the methods disclosed herein have the further advantage of identifying HLA binding peptides for HLA alleles associated with various disorders as well as alleles which are present at low frequency. Accordingly, in one method the HLA allele is present at a frequency of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% within a population. In some embodiments, the HLA allele is present at a frequency of less than 1% within a population.

[0074] In some embodiments, the methods further comprise isolating peptides from HLA- peptide complexes and sequencing the peptides to identify input candidate peptides. In someWSGR Docket No.: 64421-706.601 embodiments, the peptides are isolated from the complex by any method known to one of skill in the art, such as acid elution. In some embodiments, any suitable sequencing method may be used, including methods employing mass spectrometry, such as liquid chromatography-mass spectrometry (LC-MS or LC-MS / MS, or alternatively HPLC-MS or HPLC-MS / MS).

[0075] In some embodiments, an HLA-allele specific binding peptide sequence database comprises at least 1000 different binding peptide sequences. The methods disclosed herein may also be used to generate a database comprising the HLA-allele specific binding peptide sequences for more than one HLA-allele, for at least two different HLA-alleles, for at least five, for at least ten, fifteen, twenty, thirty or more different alleles.

[0076] In one aspect, the present disclosure provides a plurality of HLA-allele specific binding peptides, or the sequences thereof, which peptides correspond to the peptides which are presented by one specific HLA allele. More particularly, in some embodiments, an HLA- allele specific binding peptide sequence database provided is obtained by carrying out the method described herein.

[0077] Structural Features of Peptides

[0078] In one aspect, the present disclosure provides methods for identifying HLA-allele specific binding peptides, which method comprises analyzing structural features indicative of occupancy of peptides on the binding pocket of HLA alleles. In certain embodiments, the structural features are amino acid residues capable of fitting a model of peptide occupancy on the binding pocket of HLA alleles (e.g., enrichment in hydrophobicity, exposed hydrophobic surface and charges determined by the peptide's conformation within the binding pocket, as well as the size and position of the various amino acid side chains). In certain embodiments, the structural features are energetic features that are encoded not by peptide sequence, but by modeled three-dimensional structures of peptide occupancy on the binding pocket of an HLA allele (e.g., energies of attraction, repulsion, and solvation; energies of side chain and backbone hydrogen bonds; and energies and probabilities of side chain and backbone conformations) (see, e.g., Alford R F, Leaver-Fay A, Jeliazkov J R, O'Meara M J, DiMaio F P, Park H, et al. The Rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput. (2017) 13:3031-48; and Riley et al., Structure Based Prediction of Neoantigen Immunogenicity. Front Immunol. 2019 Aug. 28; 10:2047).

[0079] In certain embodiments, the binding pocket is determined by a structural analysis. Non-limiting structural analysis methods include X-ray crystallography and nuclear magneticWSGR Docket No.: 64421-706.601 resonance (NMR) spectroscopy (Alberts B, Johnson A, Lewis J, et al. Molecular Biologyof the Cell. 4th edition. New York: Garland Science; 2002. Analyzing Protein Structure and Function). In certain embodiments, a crystal structure is used to determine the binding pocket. Any HLA-allele crystal structure may be used to simulate occupancy of peptides in the binding pocket. In certain embodiments, a structure is obtained by generating a crystal structure of an HLA-allele. In certain embodiments, the crystal structures for HLA molecules are obtained from a database (e.g., the Protein Data Bank (PDB, rcsb.org)). In certain embodiments, structures of similar HLA alleles are used if a structure for the HLA-allele of interest is unavailable or cannot be generated. In certain embodiments, similar HLA-alleles include HLA alleles having the highest similarity between the amino acid sequences of the binding pocket. In some embodiments, the similarity of the binding pocket can be computed as the sum of pair- wise residue similarities according to a 20x20 amino acid similarity matrix. In certain embodiments, similar HLA-alleles include HLA alleles having similarity between the binding motifs (i.e. two alleles are considered similar if they bind similar peptides). In certain embodiments, the HLA alleles bind to two or more of the same peptides. In certain embodiments, similar HLA-alleles include HLA alleles from the same class (e.g., HL A- A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K and HLA-L). In certain embodiments, similar HLA-alleles include HLA alleles having the highest amino acid sequence identity to the HLA-allele of interest. In certain embodiments, similar HLA-alleles include HLA alleles having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the HLA-allele of interest.

[0080] In certain embodiments, structural features described herein are identified by simulating models of how the candidate peptides occupy the HLA binding pocket. In certain embodiments, the method comprises analyzing simulations of the occupancy of peptides on the binding pocket of HLA alleles.

[0081] In certain embodiments, how peptides occupy the HLA binding pocket is computationally simulated. For example, using a Multiple Sequence Comparison by Log- Expectation (MUSCLE) peptide sequence alignment, which takes into consideration positional sequence similarity, of the candidate peptide epitopes, e.g. TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1A, or PTEN peptides, with all 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17- mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes that bind each HLA and have a crystal structure available.WSGR Docket No.: 64421-706.601

[0082] In certain embodiments, the immunogenic peptides comprise from about five amino acids to about 50 amino acids. In certain embodiments, the immunogenic peptides comprise from about seven amino acids to about 45 amino acids. In certain embodiments, the immunogenic peptides comprise from about eight amino acids to about 40 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 35 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 30 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 25 amino acids.

[0083] In certain embodiments, the immunogenic peptides comprise about 5 to 50 amino acids selected from a sequence of Table 1. In certain embodiments, the immunogenic peptides comprise from about seven amino acids to about 45 amino acids selected from a sequence of Table 1. In certain embodiments, the immunogenic peptides comprise from about eight amino acids to about 40 amino acids selected from a sequence of Table 1. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 35 amino acids selected from a sequence of Table 1. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 30 amino acids selected from a sequence of Table 1. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 25 amino acids selected from a sequence of Table 1.

[0084] Table 1 below shows exemplary sequences from which immunogenic peptides may be generated.WSGR Docket No.: 64421-706.601Table 1WSGR Docket No.: 64421-706.601WSGR Docket No.: 64421-706.601WSGR Docket No.: 64421-706.601WSGR Docket No.: 64421-706.601WSGR Docket No.: 64421-706.601WSGR Docket No.: 64421-706.601

[0085] In certain embodiments, the immunogenic peptides comprise from about five amino acids to about 50 amino acids and include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at leastWSGR Docket No.: 64421-706.60189%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1. In some embodiments, an immunogenic peptide disclosed herein comprises at least a portion of the amino acid sequence of any one of the sequences of Table 1. In some embodiments, an immunogenic peptide disclosed herein comprises at least a portion of the amino acid sequence of any one of the sequences of Table 1 having one or more conserved amino acid substitutions. In certain embodiments, the immunogenic peptides comprise at least a portion of an amino acid sequence having at least 95% sequence identity to any one or more of the sequences of Table 1 having one or more conservative substitutions.

[0086] In certain embodiments, the immunogenic peptides comprise five consecutive amino acids of any one or more of the sequences of Table 1. In certain embodiments, the immunogenic peptides comprise five consecutive amino acids of any one or more of the sequences of Table 1. In certain embodiments, the immunogenic peptides comprise six consecutive amino acids of any one or more of the sequences of Table 1. In certain embodiments, the immunogenic peptides comprise seven consecutive amino acids of any one or more of the sequences of Table 1. In certain embodiments, the immunogenic peptides comprise eight consecutive amino acids of any one or more of the sequences of Table 1. In certain embodiments, the immunogenic peptides comprise nine consecutive amino acids of any one or more of the sequences of Table 1.

[0087] In certain embodiments, the immunogenic peptides comprise from about seven amino acids to about 45 amino acids and include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1

[0088] In certain embodiments, the immunogenic peptides comprise from about eight or nine amino acids to about 30 or 40 amino acids and include an amino acid sequence that has at least60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at leastWSGR Docket No. : 64421-706.60181%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1.

[0089] In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 35 amino acids and include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1

[0090] In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 30 amino acids and include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1

[0091] In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 25 amino acids and include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the sequences of Table 1WSGR Docket No.: 64421-706.601

[0092] In certain embodiments, any one of the immunogenic peptides of the present disclosure comprise at least a portion of any one of the amino acid sequences of Table 1. In certain embodiments, any one of the immunogenic peptides of the present disclosure are mutated to comprise one or more amino acid substitutions in the at least a portion of any one of the amino acid sequences of Table 1. In some embodiments, the one or more amino acid substitutions are present at the N and / or the C terminus. In some embodiments, any one of the immunogenic peptides of the present disclosure comprise one or more amino acid substitutions at one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97. 98. 99. 100. 101. 102. 103. 104. 105. 106 , 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,WSGR Docket No.: 64421-706.601535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553,554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629,630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648,649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686,687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705,706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724,725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743,744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781,782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800,801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819,820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857,858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876,877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895,896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914,915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933,934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952,953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971,972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990,991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 of any one of at least a portion of any one of the amino acid sequences of Table 1, if applicable. In some embodiments, the one or more amino acid substitutions comprise substitution of a naturally occurring amino acid with the natural, unnatural, modified, or otherwise variant version of any known amino acid including but not limited to: Alanine, Arginine, Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Selenocysteine, Pyrrolysine, Ornithine, Citrulline, Taurine, 4-Hydroxyproline, 5-Hydroxylysine, N-methylarginine, Beta- Alanine, Gamma-Aminobutyric acid (GABA), Alpha-amino-n-butyric acid, Homocysteine, D- Serine, L-Serine, D-Phenylalanine, L-Phenylalanine, O-phosphoserine, O-phosphothreonine,WSGR Docket No.: 64421-706.601Methionine sulfoxide, Methionine sulfone, N-acetylcysteine, 2-Aminobutyric acid, Sarcosine, N,N-Dimethylglycine, D-Arginine, L-Arginine, D-Tyrosine, L-Tyrosine, 2-Aminoisobutyric acid, Ornithine hydrochloride, L-Citrulline, D-Citrulline, L-Taurine, 2,4-Diaminobutyric acid, 3-Hydroxyproline, 2,3 -Diaminopropanoic acid, D-Alanine, L-Alanine, 3-Methylhistidine, Beta-Methylphenylalanine, N-(2-Aminoethyl)glycine, N-Carbamylglutamate, L- Cystathionine, 2-Methylthio-phenylalanine, 2-Aminoisobutyric acid, L-Methionine sulfoxide, 5-Formyl-2,4-dihydroxyphenylalanine, 3 -Aminopropanoic acid, 2,5-Diaminopentanoic acid, 3-Amino-2-hydroxybutyric acid, D-Glutamic acid, L-Glutamic acid, L-Histidine, D- Isoleucine, L-Leucine, D-Lysine, D-Methionine, L-Phenylalanine, D-Proline, L-Serine, D- Threonine, L-Tryptophan, D-Valine, L-Valine, L-a-Amino-y-phenylbutyric acid, any unnatural side chain-containing amino acid; a-m ethyl amino acids: e.g., a-methyl alanine, a- methyl serine; D-amino acids: e.g., D-phenylalanine, D-lysine, D-glutamic acid; P-amino acids: e.g., P-alanine, P-phenylalanine; N-methylated amino acids: e.g., N-methyl-leucine, N- methyl-arginine; azido amino acids: e.g., azidoalanine; cyclopropyl amino acids: e.g., cyclopropylglycine; fluorinated amino acids: e.g., 4-fluorophenylalanine; hydroxylated amino acids: e.g., 4-hydroxyproline; and homologated amino acids: e.g., homoserine, homophenylalanine. In some embodiments, the mutated immunogenic peptides display enhanced immunogenicity relative to their respective parental peptides. In some embodiments, the mutated immunogenic peptides display 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher or enhanced immunogenicity relative to their respective parental peptides.

[0093] In certain embodiments, mutant KRAS epitopes comprise G12V, G12D, G12C, G12R, G12A, G13D or combinations thereof.

[0094] In certain embodiments, a mutant peptide epitope comprises at least a portion of the amino acid sequence of one or more of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

[0095] In certain embodiments, the TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN parental or mutant peptides comprise one or more modified amino acids, unnatural amino acids, substituted amino acids orWSGR Docket No.: 64421-706.601 combinations thereof. Accordingly, the TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN parental or mutant peptides further comprise one or more modified amino acids, unnatural amino acids, substituted amino acids or combinations thereof. Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo- phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L- phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid containing amino acid; an a, a disubstituted amino acid; a P-amino acid; and a cyclic amino acid other than proline.

[0096] Candidate Therapeutic Peptides

[0097] In certain embodiments, the methods described herein are used to determine an effective neoantigen vaccine. In this context, it is of interest to determine which neoantigen peptides are likely to bind to a subject's HLA so as to effectively function as immunogenic peptides.WSGR Docket No.: 64421-706.601

[0098] In some embodiments, subject specific HL A alleles or HL A genotype of a subject may be determined by any method known in the art.

[0099] One of the barriers to developing curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic change (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells, represent the most tumor-specific class of antigens. Neoantigens have rarely been used in cancer vaccine or immunogenic compositions due to technical difficulties in identifying them, selecting optimized neoantigens, and producing neoantigens for use in a vaccine or immunogenic composition. These problems may be addressed by: identifying mutations in neoplasias / tumors which are present at the DNA level in tumor but not in matched germline samples from a high proportion of subjects having cancer; analyzing the identified mutations by the methods embodied herein to generate a plurality of neoantigen epitopes that are expressed within the neoplasia / tumor and that bind to a high proportion of patient HLA alleles; and synthesizing the plurality of the neoantigenic peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of subjects having cancer.

[0100] In some embodiments, a therapeutic vaccine disclosed herein may include: (1) identification of mutated peptides that can bind to HLA molecules of a high proportion of individuals and (2) formulating the drug as a multi-epitope vaccine of long peptides. In some embodiments, targeting as many mutated epitopes as possible can take advantage of the enormous capacity of the immune system, prevents the opportunity for immunological escape by down-modulation of a particular immune targeted gene product, and compensates for the known inaccuracy of epitope prediction approaches. In some embodiments, a synthetic peptide described herein provide a particularly useful means to prepare multiple immunogens efficiently and to rapidly translate identification of mutant epitopes to an effective vaccine. In some embodiments, peptides can be readily synthesized chemically and easily purified utilizing reagents free of contaminating bacteria or animal substances. The small size allows a clear focus on the mutated region of the protein and also reduces irrelevant antigenic competition from other components (unmutated protein or viral vector antigens). In some embodiments, a vaccine disclosed herein can be used in (3) combination with a strong vaccine adjuvant. In some embodiments, an effective vaccine can require a strong adjuvant to initiate an immune response. In some embodiments, Poly-ICLC, an agonist of TLR3 and the RNA helicasedomains of MDA5 and RIG3, has shown several desirable properties for a vaccine adjuvant.WSGR Docket No.: 64421-706.601These properties include the induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen-presentation by DCs. Furthermore, poly-ICLC can induce durable CD4+and CD8+responses in humans.

[0101] In certain embodiments, methods of identifying the most suitable peptides for preparing an immunogenic composition for a subject, comprises selecting from set given set of peptides the plurality of peptides capable of binding an HL A protein of the subject. In some embodiments, provided herein are methods of identifying a plurality of subject-specific peptides for preparing a subject-specific immunogenic composition, wherein the subject has a tumor and the subject-specific peptides are specific to the subject and the subject's tumor. In certain embodiments, the subject-specific peptides, each comprise a different tumor neoepitope that is an epitope specific to the tumor of the subject and each binds an HLA protein of the subject, as provided by the methods for identifying HLA binding described herein.

[0102] In certain embodiments, the cell used in the method for determining HLA binding as described herein is an antigen-presenting cell.

[0103] In some embodiments, T cell receptors which recognize peptide-HLA combinations as disclosed herein are identified. It is contemplated that unique T cell receptors are characterized which recognize particular peptide-HLA combinations. In some embodiments, the unique T cell receptors recognize specific heteroclitic neoepitope complexes. In some embodiments, the unique T cell receptors recognize specific heteroclitic neoepitope complexes with enhanced immunogenicity.

[0104] Neoantigens

[0105] In certain embodiments, the tumor antigens that bind to HLA alleles are identified by the methods described herein. In certain embodiments, the tumor antigens are neoantigens. In a further aspect, the disclosure provides methods for identifying tumor neoantigen-comprising peptides (i.e., neoepitope, neoantigenic peptide), wherein the methods comprise identifying for a given HLA allele, the peptides binding an HLA allele in a tumor cell from a tumor of a patient.

[0106] In some embodiments, the tumor antigen comprises tumor protein 53 (TP53), isocitrate dehydrogenase 1 (IDH1), catenin beta 1 (CTNNB1), epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2), Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma oncogene homolog Bl (BRAF), F-box and WD repeat domain containing 7 (FBWX7), fibroblast growth factor receptor 3 (FGFR3), mucin 4 (MUC4),WSGR Docket No.: 64421-706.601 neuroblastoma RAS viral oncogene homolog (NRAS), phosphatidylinositol-4,5-bisphosphate 3 -kinase catalytic subunit alpha (PIK3CA), protein phosphatase 2 regulatory subunit A alpha (PPP2R1A), or phosphatase and tensin homolog (PTEN). In some embodiments, the tumor antigen comprises one or more mutations. In some embodiments, the one or more mutations comprises TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTENR130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

[0107] In some embodiments, mutated epitopes are effective in inducing an immune response. In some embodiments, spontaneous tumor regression or long-term survival correlate with CD8+T-cell responses to mutated epitopes (Buckwaiter and Srivastava P K. “It is the antigen(s), stupid” and other lessons from over a decade of vaccine therapy of human cancer. Seminars in immunology 20:296-300 (2008); Karanikas el al., High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival. Cancer Res. 61 :3718-3724 (2001); Lennerz et al., The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci USA. 102: 16013 (2005)) and that “immunoediting” can be tracked to alterations in expression of dominant mutated antigens in mice and man (Matsushita et al., Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting Nature 482:400 (2012); DuPage et al., Expression of tumor-specific antigens underlies cancer immunoediting. Nature 482:405 (2012); and Sampson et al., Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol. 28:4722- 4729 (2010)).

[0108] In some embodiments, each tumor contains multiple, patient-specific mutations that alter the protein coding content of a gene. Such mutations create altered proteins, ranging from single amino acid changes (caused by missense mutations) to addition of long regions of novel amino acid sequence due to frame shifts, read-through of termination codons or translation of intron regions (novel open reading frame mutations; neoORFs). In some embodiments, these mutated proteins are valuable targets for the host's immune response to the tumor as, unlike native proteins, they are not subject to the immune-dampening effects of self-tol erance. In someWSGR Docket No.: 64421-706.601 embodiments, mutated proteins are more likely to be immunogenic and are also more specific for the tumor cells compared to normal cells of the patient. The mutated proteins can be referred to as neoantigens. The term “neoantigen” or “neoantigenic” means a class of tumor antigens that arises from a tumor-specific mutation(s) which alters the amino acid sequence of genome encoded proteins. In some embodiment, the mutated tumor antigen induces a higher level of immune response as compared to a corresponding parental or wildtype tumor antigen.

[0109] Embodiments disclosed herein provide a method of identifying peptides, e.g., neoantigens, including, but not limited to novel unannotated open reading frames (nuORFs), that are capable of eliciting a cancer specific T-cell response. Genomic aberrations in cancer cells give rise to mutant peptides (neoantigens) displayed on the human leukocyte antigen (HLA) molecules and recognized by T cells, thus triggering an immune response against cancer cells. In some embodiments, patients vaccinated with neoantigen-based peptides can display expanded neoantigen-specific T cells, and is a promising avenue for cancer treatment (Ott et al., An immunogenic personal neoantigen vaccine or patients with melanoma, Nature 2017 Jul. 13; 547(7662):217-221; Sahin et al., 2017 Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer”, Nature, vol. 547, 2017, pages 222-226). In some embodiments, neoantigens can be predicted based on mutations detected by whole exome sequencing (WES). In some embodiments, their expression levels are estimated using mRNA sequencing (RNA-seq). Ribosome profiling (Ribo-seq) allows to monitor mRNA translation and has been used to predict a plethora of translated novel unannotated ORFs (nuORFs) (Fields et al., 2015. A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation, Mol Cell, vol. 60, pages 816- 827; Ji et al., 2015. Many IncRNAs, 5'UTRs, and pseudogenes are translated and some are likely to express functional proteins, ELIFE, vol. 4). Ribo-seq analysis of human fibroblasts infected with HSV-1 and HCMV has identified nuORFs that contribute peptides presented on major histocompatibility complex class I (MHC I) (Erhard et al., 2018. Improved Ribo-seq enables identification of cryptic translation events, Nat. Methods, vol. 15, no. 5, pages 363- 366).

[0110] The methods disclosed herein can be used to select subject specific peptides that are presented by a tumor for any neoplasia. By “neoplasia” is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a neoplasia. Examples of cancers include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acuteWSGR Docket No.: 64421-706.601 myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0111] Vaccines and Immunological Compositions

[0112] In certain embodiments, the peptides identified according to the present disclosure are used in a vaccine or immunological composition to treat any disease or condition described herein (e.g., tumor, autoimmunity, infection, transplant). The term “vaccine” or “immunological composition” are used interchangeably and are meant to refer in the present context to a pooled sample of one or more antigenic peptides, for example at least one, at least two, at least three, at least four, at least five, or more antigenic peptides. A “vaccine” is to be understood as including a protective vaccine, which is a composition for generating immunity for the prophylaxis and / or treatment of diseases (e.g., neoplasia / tumor). A “vaccine” is also to be understood as including a tolerizing vaccine, which is a composition for reducing immunity for the prophylaxis and / or treatment of diseases (e.g., autoimmune disease). A tolerizing vaccine may be formulated with antigenic epitopes specific for an allergen or for an autoimmunity antigen identified according to the present disclosure. In some embodiments, a protective vaccine may be formulated with antigenic epitopes specific for a pathogen or for a cancer cell. Accordingly, In some embodiments, vaccines can be medicaments which compriseWSGR Docket No.: 64421-706.601 antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination. A “vaccine composition” can include a pharmaceutically acceptable excipient, carrier or diluent.

[0113] In some embodiments, the vaccine may include one or more peptides identified according to the present disclosure. For example, 1 to 10 peptides. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0114] In certain embodiments, a protective vaccine is used to treat cancer. Additional examples of cancers and cancer conditions that can be treated with the therapy of this document include, but are not limited to, a patient in need thereof that has been diagnosed as having cancer, or at risk of developing cancer. The subject may have a solid tumor such as breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs and hematological tumors, such as lymphomas and leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B cell lymphomas, tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, such as glioblastomas or medulla blastomas); head and / or neck cancer, breast tumors, tumors of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); tumors of the blood and lymphatic system’ (e.g., Hodgkin's disease , Non-Hodgkin's disease lymphoma, Burkitt's lymphoma, AIDS-related lymphomas, malignant immunoproliferative diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell type, leukemia of unspecified cell type, unspecified malignant neoplasms of lymphoid, hematopoietic and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma); tumors of the excretory system (e.g., kidney, renal pelvis, ureter, bladder, and other urinary organs); tumors of the gastrointestinal tract (e.g.,WSGR Docket No.: 64421-706.601 esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal); tumors involving the liver and intrahepatic bile ducts, gall bladder, and other parts of the biliary tract, pancreas, and other digestive organs; tumors of the oral cavity (e.g., lip, tongue, gum, floor of mouth, palate, parotid gland, salivary glands, tonsil, oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites of the oral cavity); tumors of the reproductive system (e.g., vulva, vagina, Cervix uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); tumors of the respiratory tract (e.g., nasal cavity, middle ear, accessory sinuses, larynx, trachea, bronchus and lung, such as small cell lung cancer and non-small cell lung cancer); tumors of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other sites); tumors of the skin (e.g., malignant melanoma of the skin, nonmelanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneoum and peritoneum, eye, thyroid, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites. Thus the population of subjects described herein may be suffering from one of the above cancer types. In other embodiments, the population of subjects may be all subjects suffering from solid tumors, or all subjects suffering from liquid tumors.

[0115] In some embodiments, cancers that can be treated using the therapy described herein may include, among others, cases which are refractory to treatment with other chemotherapeutics. The term “refractory, as used herein refers to a cancer (and / or metastases thereof), which shows no or only weak antiproliferative response (e.g., no or only weak inhibition of tumor growth) after treatment with another chemotherapeutic agent. These are or can be cancers that cannot be treated satisfactorily with other chemotherapeutics. In some embodiments, refractory cancers encompass not only (i) cancers where one or more chemotherapeutics have already failed during treatment of a patient, but also (ii) cancers that can be shown to be refractory by other means, e.g., biopsy and culture in the presence of chemotherapeutics.

[0116] In some embodiments, the therapy described herein is also applicable to the treatment of patients in need thereof who have not been previously treated.WSGR Docket No.: 64421-706.601

[0117] In some embodiments, the therapy described herein is also applicable where the subject has no detectable neoplasia but is at high risk for disease recurrence.

[0118] In some embodiments, the therapy described herein is also applicable where the subject has undergone Autologous Hematopoietic Stem Cell Transplant (AHSCT), and in particular patients who demonstrate residual disease after undergoing AHSCT. The post- AHSCT setting is characterized by a low volume of residual disease, the infusion of immune cells to a situation of homeostatic expansion, and the absence of any standard relapse-delaying therapy. These features provide a unique opportunity to use the neoplastic vaccine or immunogenic composition compositions to delay disease relapse.

[0119] In Vitro Peptide / Polypeptide Synthesis

[0120] Proteins or peptides disclosed herein may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides. In some embodiments, the nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases located at the National Institutes of Health website. The coding regions for known genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.

[0121] In some embodiments, peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield R B: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). In certain embodiments, neoantigenic peptides are prepared by (1) parallel solid-phase synthesis on multi-channel instruments using uniform synthesis and cleavage conditions; (2) purification over a RP-HPLC column with column stripping; and re-washing, but not replacement, between peptides; followed by (3) analysis with a limited set of the most informative assays. The Good Manufacturing Practices (GMP) footprint can be defined around the set of peptides for an individual patient, thus requiring suite changeover procedures only between syntheses of peptides for different patients.WSGR Docket No.: 64421-706.601

[0122] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding a neoantigenic peptide of the disclosure may be used to produce the neoantigenic peptide in vitro. The polynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single- and / or double-stranded, or native or stabilized forms of polynucleotides, such as e.g. polynucleotides with a phosphorothioate backbone, or combinations thereof and it may or may not contain introns so long as it codes for the peptide. In some embodiments, in vitro translation is used to produce the peptide. Many exemplary systems exist that one skilled in the art could utilize (e.g., Retie Lysate IVT Kit, Life Technologies, Waltham, Mass.).

[0123] An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into the host bacteria for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.).

[0124] Expression vectors comprising the isolated polynucleotides, as well as host cells containing the expression vectors, are also contemplated. The neoantigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides. One or more neoantigenic peptides of the disclosure may be encoded by a single expression vector.

[0125] In certain embodiments, the polynucleotides may comprise the coding sequence for the tumor specific neoantigenic peptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and / or secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.

[0126] In certain embodiments, isolated nucleic acid molecules having a nucleotide sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80%) identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%), 97%, 98% or 99% identical to a polynucleotide encoding a tumor specificWSGR Docket No.: 64421-706.601 neoantigenic peptide of the present disclosure, can be provided. By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the amino- or carboxy-terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0127] As a practical matter, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present disclosure, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% > of the total number of nucleotides in the reference sequence are allowed.

[0128] Recombinant expression vectors may be used to amplify and express DNA encoding the tumor specific neoantigenic peptides. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a tumor specific neoantigenic peptide or a bioequivalent analog operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, a transcriptional unit comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promotersWSGR Docket No.: 64421-706.601 or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail herein. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) can be operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter can be operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site can be operatively linked to a coding sequence if it is positioned so as to permit translation. Generally, operatively linked means contiguous, and in the case of secretory leaders, means contiguous and in reading frame. Structural elements intended for use in yeast expression systems can include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0129] Useful expression vectors for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising expression control sequences from SV40, bovine papillomavirus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as MI 3 and filamentous single-stranded DNA phages.

[0130] Suitable host cells for expression of a polypeptide include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

[0131] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified andWSGR Docket No.: 64421-706.601 completely functional. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0132] The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography, and the like), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S-transferase, and the like can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0133] In Vivo Peptide / Polypeptide Synthesis

[0134] The present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering neoantigenic peptides / polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA / RNA vaccines.

[0135] In certain embodiments, antigens may be administered to a patient in need thereof by use of a plasmid. In some embodiments, these can be plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995), Journal Immunol. 155 (4): 2039-2046). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression (Leitner et al. (1997), Journal Immunol. 159 (12): 6112-6119). Plasmids also include a strong polyadenylation / transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences Multi cistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.WSGR Docket No.: 64421-706.601

[0136] In some embodiments, a way of enhancing protein expression is by optimizing the codon usage of pathogenic mRNAs for eukaryotic cells. Another consideration is the choice of promoter. Such promoters may be the SV40 promoter or Rous Sarcoma Virus (RSV). Plasmids may be introduced into animal tissues by a number of different methods. The two most used approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. In some embodiments, injection in saline can be conducted intramuscularly (EVI) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation by temporarily damaging muscle fibers with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose. Immune responses to this method of delivery can be affected by many factors, including needle type, needle alignment, speed of injection, volume of injection, muscle type, and age, sex and physiological condition of the animal being injected. In some embodiments, DNA or RNA that encodes a peptide disclosed herein can be administered to a subject.

[0137] Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA may also be delivered to cells following mild mechanical disruption of the cell membrane, temporarily permeabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Ex vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, Sharei et al., PLOS ONE DOI: 10.1371 / joumal.pone.Ol 18803 Apr. 13, 2015).

[0138] In certain embodiments, a neoplasia vaccine or immunogenic composition may include separate DNA plasmids encoding, for example, one or more neoantigenic peptides / polypeptides as identified in according to the disclosure. As discussed herein, the exact choice of expression vectors can depend upon the peptide / polypeptides to be expressed and is well within the skill of the ordinary artisan. The expected persistence of the DNA constructs (e.g., in an episomal, non-replicating, non-integrated form in the muscle cells) is expected to provide an increased duration of protection.

[0139] One or more antigenic peptides of the disclosure may be encoded and expressed in vivo using a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the neoplasia vaccine or immunogenicWSGR Docket No.: 64421-706.601 composition may include a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis. 2013 Jan. 15; 207(2):240-7). Plasmids that can be used for adeno associated virus, adenovirus, and lentivirus delivery have been described previously. The peptides and polypeptides of the disclosure can also be expressed by a vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or lentivirus. In some embodiments, this approach can involve the use of a vector to express nucleotide sequences that encode the peptide of the disclosure. Upon introduction into an acutely or chronically infected host or into a noninfected host, the vector can express the immunogenic peptide, and thereby elicits a host CTL response.

[0140] Among vectors that may be used in the practice of the disclosure, integration in the host genome of a cell is possible with retrovirus gene transfer methods, often resulting in long term expression of the inserted transgene. In certain embodiments, the retrovirus is a lentivirus. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus. In some embodiments, cell type specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and hence both lentiviral and retroviral vectors may be used in the practice of the disclosure). Moreover, In some embodiments, lentiviral vectors can be preferred as they are able to transduce or infect non-dividing cells and may produce high viral titers. Selection of a retroviral gene transfer system may therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the desired nucleic acid into the target cell to provide permanent expression. Widely used retroviral vectors that may be used in the practice of the disclosure include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.WSGR Docket No.: 64421-706.601

[0141] Also useful is a minimal non-primate lentiviral vector, such as a lentiviral vector based on the equine infectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med, 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / jgm.845). The vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene. Accordingly, the disclosure contemplates amongst vector(s) useful in the practice of the disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[0142] One of skill in the art can determine suitable dosage. Suitable dosages for a virus can be determined empirically. Also useful in the practice of the disclosure is an adenovirus vector. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in a variety of mammalian cells and tissues in vitro and in vivo, resulting in the high expression of the transferred nucleic acids. Further, the ability to productively infect quiescent cells, expands the utility of recombinant adenoviral vectors. In addition, high expression levels ensure that the products of the nucleic acids will be expressed to sufficient levels to generate an immune response (see e.g., U.S. Pat. No. 7,029,848).

[0143] In some embodiments, an adenovirus vector used can be selected from the group consisting of the Ad5, Ad35, Adi 1, C6, and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”) genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285). Ad35 vectors are described in U.S. Pat. Nos. 6,974,695, 6,913,922, and 6,869,794. Adenovirus vectors that are El-defective or deleted, E3- defective or deleted, and / or E4-defective or deleted may also be used. Certain adenoviruses having mutations in the El region have improved safety margin because El-defective adenovirus mutants are replication-defective in non-permissive cells, or, at the very least, are highly attenuated. Adenoviruses having mutations in the E3 region may have enhanced the immunogenicity by disrupting the mechanism whereby adenovirus down-regulates MHC class I molecules. Adenoviruses having E4 mutations may have reduced immunogenicity of the adenovirus vector because of suppression of late gene expression. Such vectors may be particularly useful when repeated re-vaccination utilizing the same vector is desired. Adenovirus vectors that are deleted or mutated in El, E3, E4, El and E3, and El and E4 can be used in accordance with the present disclosure. Furthermore, “gutless” adenovirus vectors, in which all viral genes are deleted, can also be used in accordance with the present disclosure. In some embodiments, such vectors require a helper virus for their replication and require aWSGR Docket No.: 64421-706.601 special human 293 cell line expressing both El a and Cre, a condition that does not exist in natural environment. Such “gutless” vectors are non-immunogenic and thus the vectors may be inoculated multiple times for re-vaccination. The “gutless” adenovirus vectors can be used for insertion of heterologous inserts / genes such as the transgenes of the present disclosure and can even be used for co-delivery of a large number of heterologous inserts / genes.

[0144] In one embodiment, the viral vector is an adenovirus vector, an adeno-associated viral vector (AAV), or derivatives thereof. The adeno-associated viral vector comprises AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, DJ / 8 or pseudotypes thereof. In one embodiment, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. In some embodiments, one can select the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. In some embodiments, AAV8 can be useful for delivery to the liver.

[0145] In another embodiment effectively activating a cellular immune response for a neoplasia vaccine or immunogenic composition can be achieved by expressing the relevant antigens in a vaccine or immunogenic composition in a non-pathogenic microorganism. Well- known examples of such microorganisms include but are not limited to Mycobacterium bo s BCG, Salmonella and Pseudomona (See, U.S. Pat. No. 6,991,797). In another embodiment a Poxvirus is used in the neoplasia vaccine or immunogenic composition. These include orthopoxvirus, avipox, vaccinia, MV A, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardi et al., Hum Vaccin Immunother. 2012 July; 8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220-4222).

[0146] In some embodiments, poxviruses that may be used in the practice of the disclosure, such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC, NYVAC. 1, NYVAC.2, MVA, MVA-BN), canarypoxvirus (e.g., Wheatley C93 Strain, ALVAC), fowlpoxvirus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non-naturally occurring recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and patent literature.

[0147] In one embodiment recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof. Dosages of expressed neoantigen canWSGR Docket No.: 64421-706.601 range from a few to a few hundred micrograms, e.g., 5 to 500 pg. The vaccine or immunogenic composition can be administered in any suitable amount to achieve expression at these dosage levels. The viral particles can be administered to a patient in need thereof or transfected into cells in an amount of about at least 103pfu; thus, the viral particles are preferably administered to a patient in need thereof or infected or transfected into cells in at least about 104pfu to about 106pfu; however, a patient in need thereof can be administered at least about 108pfu or at least about 107pfu to about 109pfu. Doses as to NYVAC are applicable as to ALVAC, MV A, MVA- BN, and avipoxes, such as canarypox and fowlpox.

[0148] Pharmaceutical Compositions / Methods of Delivery

[0149] In certain embodiments, a pharmaceutical composition comprises an effective amount of one or more antigenic peptides as described herein (including a pharmaceutically acceptable salt, thereof), optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.

[0150] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0151] A “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0152] A “pharmaceutically acceptable salt” of pooled tumor specific neoantigens as recited herein may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC — (CFBjn-COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are notWSGR Docket No.: 64421-706.601 limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceutically acceptable salts for the pooled tumor specific neoantigens provided herein, including those listed by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

[0153] When administered as a combination, the therapeutic agents, for example, the neoantigenic peptides, can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

[0154] The compositions may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year. The dosing interval can be adjusted according to the needs of individual patients. For longer intervals of administration, extended release or depot formulations can be used.

[0155] Combination Therapies

[0156] The disclosure also contemplates the combination of the composition of the present disclosure with other drugs and / or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. For example, in the case of cancer chemotherapeutic agents may be administered as part of the combination therapy.

[0157] In certain embodiments, the TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN peptides are administered in conjunction with a cancer therapy. In some embodiments, the compositions disclosed herein are administered in conjunction with a cancer therapy. As used herein, the term “cancer therapy” refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, chemotherapeutic agents, immunotherapy,WSGR Docket No.: 64421-706.601 growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti -tubulin agents, and other agents to treat cancer, such as anti-HER- 2 antibodies (e.g., HERCEPTIN™), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVA™)), platelet derived growth factor inhibitors (e.g., GLEEVEC™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s), TRAIL / Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also contemplated for use with the methods described herein.

[0158] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA™, Genentech / OSI Pharm.), Bortezomib (VELCADE™, Millennium Pharm.), Fulvestrant (FASLODEX™, Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA™, Novartis), Imatinib mesylate (GLEEVEC™, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin™, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE™, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA™, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXAN™ cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozcicsin, carzcicsin and bizcicsin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin yl and calicheamicin omega 1 (Angew Chem. Inti. Ed. Engl. (1994) 33: 183-WSGR Docket No.: 64421-706.601186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, strcptonigrin, strcptozocin, tubcrcidin, ubenimcx, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; tr iaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR™ gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;WSGR Docket No.: 64421-706.601 difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0159] Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX™ (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON™ (toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane), formestanie, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole), and ARIMIDEX™ (anastrozole); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME™ (ribozyme)) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, and VAXID™ vaccine; PROLEUKIN™ rIL-2; LURTOTECAN™ topoisomerase 1 inhibitor; ABARELIX™ rmRH; (x) anti -angiogenic agents such as bevacizumab (AVASTIN™, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0160] In various embodiments, the cancer therapeutic is an immunotherapy selected from the group comprising oncolytic virus, bacteria, oncolytic bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), a microbiome modulator, and / or a toll-like receptor (TLR) agonist. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonist. In various embodiments, the TLR agonist is derived from virus, plants, bacteria and / or made synthetically. In various embodiments, the immunotherapy is a is a stimulator of interferon genes (STING) pathway modulator.

[0161] It will be appreciated by those skilled in the art of cancer immunotherapy that other complementary immune therapies may be added to the regimens described above to further enhance their efficacy including but not limited to GM-CSF to increase the number of myeloid derived innate immune system cells, low dose cyclophosphamide or PI3K inhibitors (e.g., PI3KWSGR Docket No.: 64421-706.601 delta inhibitors) to eliminate T regulatory cells that inhibit innate and adaptive immunity and 5FU (e.g., capecitabine), PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells. For example, PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC- 0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TGI 00-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some aspects, the PI3K inhibitor is aPI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503. The immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INFa.

[0162] In certain embodiments, TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFRL858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1 A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, and TP53 R241Y peptides are administered with one or more immune checkpoint modulators. In some embodiments, the composition disclosed herein can be administered with an immune checkpoint modulator. Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses. Examples of checkpoint inhibitor include but are not limited to an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-p.

[0163] The term “checkpoint inhibitor” means a group of molecules on the cell surface of CD4+and / or CD8+T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPa (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012 / 177624). “Anti-immune checkpoint inhibitor therapy” refers to the use of agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. Exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, smallWSGR Docket No.: 64421-706.601 molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and / or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and / or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-Ll antibodies, and anti-CTLA-4 antibodies, either alone or used in combination.

[0164] In some embodiments, such therapy involves blockade of programmed cell death 1 (PD-1). In some embodiments, such therapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1). In some embodiments, such therapy involves administration of an antibody agent that specifically interacts with PD-1 or with PD- Ll. In some embodiments, such therapy involves administration of one or more of nivolumab (BMS-936558, MDX-1106, ONO-4538, a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, a humanized monoclonal IgG4 anti- PD-1 antibody), BMS-936559 (a fully human IgG4 PD-L1 antibody), MPDL3280A (a humanized engineered IgGl monoclonal PD-L1 antibody) and / or MEDI4736 (a humanized engineered IgGl monoclonal PD-L1 antibody).WSGR Docket No.: 64421-706.601

[0165] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the disclosure.

[0166] This disclosure is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures and the sequence listing, are hereby incorporated by reference.EXAMPLES

[0167] EXAMPLE 1: HETEROCLITIC NEOEPITOPE VACCINES DERIVED FROM COMPUTATIONAL STRUCTURAL MODELING

[0168] A library of KRAS mutants, TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, and TP53 R241Y heteroclitic epitopes is generated for structural analysis in a panel of HLAs representing >90% of the global population. For example, the consensus binding motif for KRAS G12D epitopes of each HLA showed distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors (FIG. 1). In some cases, major anchor residues were found predominately at position 2, 3, 5, and 9. Some alleles have additional minor anchor residues at positions 1, 4, 6, and 7.

[0169] Using, for example, KRAS mutants, TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, and TP53 R241 Y 9-mer epitopes, a library of amino acid modified peptides is generated with every combination of anchor residue amino acids specific for each HLA subgroup. As proof of concept, NetMHC prediction of heteroclitic epitope binding affinity within each HLA subgroup is performed in silico and indicates an increase in binding affinity in some epitopes for all HLA types.WSGR Docket No.: 64421-706.601

[0170] To demonstrate the structural modelling approach herein, each TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y peptide is modelled onto one or more of HLA-A*02:01, HLA-A*03:01, HLA- A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 to investigate peptide-HLA interactions. A Multiple Sequence Comparison by Log-Expectation (MUSCLE) peptide sequence alignment is performed which takes into consideration positional sequence similarity of the TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y peptide epitopes with all epitopes that bind each HLA and have a crystal structure available. Using the HLA- peptide structure with the greatest sequence similarity, the TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y peptide neoepitopes are modeled into the HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position. The resulting HLA-peptide neoepitope complexes are structurally validated using the root-mean-square deviation analysis (Ca- RMSD) to ensure no major destabilizing structural changes have occurred to overall HLA cleft. High-resolution Monte Carlo with minimization docking of the peptide to the HLA cleft is performed to identify the lowest full-atom energy conformation. The energetic-optimized structures are used to introduce each anchor residue modification found in the heteroclitic epitope library. Using this approach, an increase in the number of contacts between peptides and HLA molecules is found after each amino acid anchor residue substitution at for example position 2, 6, and 9, demonstrating the ability to detect structural interaction differences in HLA-heteroclitic peptide interactions.WSGR Docket No.: 64421-706.601

[0171] HLA-specific mutant TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1 A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y peptide minimal epitope targets are validated by mass-spectrometry for heterolytic peptide design. In one instance, HLA alleles of interest for KRAS mutants were validated and are noted in FIG. 2.

[0172] Neoepitope-HLA structural models of the present disclosure are used to analyze the stereochemical changes between parental peptides and modified peptides. Structural parameters are analyzed such as contacts between peptide and HLA cleft residues, solvent- assessable surface area (SAS A), peptide rigidity as a measure of CaRMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, and electrostatic potential. These measurements are compared to modelled parental neoepitopes in each HLA molecule.

[0173] Using the neoepitope-HLA structural models, amino acid modifications of modified TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTENR130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y peptide or APL epitopes with improved HLA binding affinity are predicted, for example for HLA-A*02:01, HLA-A*03:01, HLA-A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03.

[0174] The predicted binding affinity (mM) of altered peptide ligands (APL) of parental and modified peptides for combinations of 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, and 25-mers and APLs to for example, HLA-A*02:01, HLA-A*03:01, HLA- A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 is generated.

[0175] The binding of particular parental and modified APLs of 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers,WSGR Docket No.: 64421-706.60121-mers, 22-mers, 23-mers, 24-mers, and 25-mers and APLs to, for example HLA-A*02:01, HLA-A*03:01, HLA-A*l l:01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA- C*01 :02, HLA-C*08:02, and HLA-C*03:03 is tested using MBL tetramer quicks witch assay. HLA tetramers are incubated with 10 mM parental or modified TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241 Y peptide epitopes at room temperature for 4 hours. Peptide loading is quantified by flow cytometry analysis with anti-exiting peptide antibody. % APL binding relative to parental peptide binding to tetramer are calculated. NetMHC prediction is used to predict binding affinities for various HLA*-specific peptides and APLs tested with this assay.

[0176] Percent binding of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFRL858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1 A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes that bind HLA molecules. In certain embodiments, the 8-mer, 9-mer, 11- mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer 21 -mer, 22- mer, 23-mer, 24-mer, or 25-mer parental epitopes and altered peptide ligands is determined.

[0177] The ability of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1 A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y APLs to prime T cell responses against parental antigen is tested in vitro. An in vitro immunogenicity assay is conducted for TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R,WSGR Docket No.: 64421-706.601PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1 A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y altered peptide ligands in, for example HLA-A*02:01, HLA-A*03:01, HLA- A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 healthy donors. Healthy donor dendritic cells are generated by adherence of monocytes to flasks and differentiated in 800U / mL hGMCSF and 400IU / mL hIL-4 for 7 days. Dendritic cells are then matured in hIL-lB, IL-6, TNFa, and PGE2 for 2 days. Mature dendritic cells are then collected and pulsed with 40 pg / mL parental or altered peptide ligand for 2hrs. Pulsed dendritic cells are washed twice with PBS and then co-cultured with autologous CD8+T cells in the presence of hIL-15, hIL-6, and hIL-21. 7 days later, more peptide-pulsed dendritic cells are added to each co-culture. The following day, fresh hIL2 and IL7 are added. 7 days post the final dendritic cell culture, T cells are collected and stained with an HLA-A*02:01, HLA-A*03:01, HLA-A*l l:01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA- C*01 :02, HLA-C*08:02, or HLA-C*03:03 tetramer containing the parental epitope sequence. Flow cytometry is used to determine T cell expansion. No tetramer control is used as a negative control.

[0178] TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTENR130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y parental and altered peptide ligand-HLA contacts and surface structure is determined. Hydrophobicity of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y parental and altered peptide ligand-HLA is measured. Electrostatic potential of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53WSGR Docket No.: 64421-706.601R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y parental and altered peptide ligand is measured. Structural feature measurements of TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTENR130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15- mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes that bind HLA molecules. In certain embodiments, the 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer 21 -mer, 22-mer, 23-mer, 24-mer, and 25-mer parental epitope and altered peptide ligands modeled in the context of HLA-A*02:01, HLA-A*03:01, HLA-A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA- B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 are determined.

[0179] Binding and immunogenicity assays are carried out for epitope and HLA combinations as disclosed herein. It is determined that certain engineered antigens show superior binding and T cell responses. Non-linear regression curves and EC50s are generated to assess binding of engineered antigens. Expanded CD8 T cells from no peptide, irrelevant peptide, or parental peptide for the peptides disclosed herein are seeded in an ELISpot capture plate coated with anti-human fFNy antibody. T cells are stimulated with control or parental antigen overnight and IFNy capture is read out. Two-way ANOVA followed by uncorrected Fischer’s LSD test is performed, (ns = P > 0.05, *P < 0.05, **P < 0.01, *** < 0.001, ****P < 0.0001). CD8 T cells expanded with certain engineered antigens show increased fFNy spots as compared to the parental control, indicating superior T cell response.

[0180] In vitro immunogenicity testing of for example HLA-A*02:01, HLA-A*03 :01, HLA- A*l l:01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, or HLA-C*03:03 parental epitopes and their engineered counterparts is carried out. Human monocyte-derived DCs (moDCs) differentiated from healthy donor PBMCs are pulsed with 100 pM parental or engineered antigen plus P2-microglobulin (4 hours) at 37°C. After peptide pulsing, moDCs are washed with PBS and co-cultured with 6>< 106autologous CD8 T cells (plus IL-2, IL-7, IL-15, and IL-21) (7 days). Boosts are performed with peptide-pulsed moDCs on day 7, 14, and 21. T cells are collected and stained with HLA-A*02:01, HLA-A*03:01,WSGR Docket No.: 64421-706.601HLA-A*11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01 :02, HLA-C*08:02, or HLA-C*03:03 tetramer loaded with parental peptide or were subject to IFNy ELISpot.

[0181] Structural analysis of engineered antigens is used to identify peptide rigidity and stabilizing HLA cleft interactions as features associated with enhanced peptide immunogenicity. Top 10 energetically favorable models are determined for each peptide. CaRMSD is calculated for the 10-mer peptide in each model compared to all other models for that epitope. Two way ANOVA is performed followed by Tukey’s multiple comparisons test. Structural analysis is carried out of peptide neoantigen residues in contact with HLA cleft residues that are associated with stabilization of peptide-HLA interactions (including but not limited to: Argl 14, Glnl55, Gln70, TRP147, and Thr73). Quantification of engineered antigen residue interactions with each of the HLA-cleft residues across the top 10 structural models for parental peptides demonstrates altered patterns of HLA cleft binding with a significantly higher proportion of interactions with for example the Gln70 residue, suggesting more diverse stabilizing interactions within the HLA cleft.

[0182] Binding affinity changes for TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, JAK2 V617F, KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, and TP53 R241Y APLs in the context of for example HLA-A*02:01, HLA-A*03:01, HLA-A* 11 :01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 are determined. Optimized APLs for HLA-A*02:01, HLA-A*03:01, HLA-A*l l:01, HLA-A*30:01, HLA-A*68:01, HLA-B*07:02, HLA-C*01:02, HLA-C*08:02, and HLA-C*03:03 are designed and tested for enhanced binding affinity relative to parental neoantigen in K562-TAP1KO-HLA expressing cell lines. Cells are incubated with increasing concentration of peptide overnight at 37C. Cells are then stained for HLA-ABC expression on the surface of cells. Median florescent intensity of HLA expression is quantified for each peptide at each peptide concentration in duplicates. Control peptide corresponds to a positive control peptide specific to each HLA. Non-linear regression curves are fit to each graph that is generated.

[0183] Immunogenicity studies as described herein allow for the identification of T cell receptors which recognize certain peptide-HLA combinations. It is contemplated that unique T cell receptors are characterized which recognize particular peptide-HLA combinations. InWSGR Docket No.: 64421-706.601 some embodiments, the unique T cell receptors recognize specific heteroclitic neoepitope complexes. In some embodiments, the unique T cell receptors recognize specific heteroclitic neoepitope complexes with enhanced immunogenicity.

[0184] Altered Peptide Ligands (APL)

[0185] Using a Panc02 murine model, altered peptide ligands (APL) were engineered or included in a murine neoantigen-targeted vaccine (H. L. Kinkead et al., J CI Insight. 2018;3(20):el22857. Doi.org / 10.1172 / jci.insight.122857). FIG. 3A-FIG. 3F demonstrate that engineered peptides display enhanced contact points and solvent accessible surface area, and reduced rigidity in a murine PDAC model. The parental peptide comprises the sequence of GSSAEESHLSGLNWSTLVPL (SEQ ID NO: 75). The APL comprises the sequence of GSSAEESHLSCLNWSTLVPL (SEQ ID NO: 76). Mice were vaccinated twice 7 days apart with PBS, 50 pg APL, or 5 pg parental peptide. Seven days after the last vaccine dose, splenocytes were restimulated with APL or parental peptides, overnight, and IFNy production was measured by ELISpot. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed (FIG 3A). FIG. 3B illustrates the in silico structural modeling of APL or parental peptide in murine H2-Kb. The number of contacts between MHC binding cleft and peptide (FIG. 3C) or mutated residue (FIG. 3D) are shown. FIG. 3E shows Ca-RMSD, measure of peptide rigidity. FIG. 3F shows the solvent accessible surface area (SASA). Unpaired students t test was performed, (ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001,

[0186] Results shown in FIGS. 4A-FIG. 4B demonstrate that KRAS G12D APL1- HLA* 11 :01 binding and immunogenicity of engineered antigens show superior binding and T Cell responses. l * 106expanded CD8 T cells from no peptide, irrelevant peptide, parental peptide, or APL1 were seeded in an ELISpot capture plate coated with anti -human IFNy antibody. FIG. 4A shows non-linear regression curves and calculated EC50s for binding between HLA-A* 11 :01 and a parental KRAS G12D 10-mer peptide (SEQ ID NO: 11) and APL (SEQ ID NO: 12).

[0187] 1 x lO6expanded CD8 T cells treated with no peptide, irrelevant peptide, parental peptide, or APL were seeded in an ELISpot capture plate coated with anti-human IFNy antibody. T cells were stimulated with control or parental antigen overnight and IFNy capture was read out. Two-way ANOVA followed by uncorrected Fischer’s LSD test was performed. (ns = P > 0.05, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). CD8 T cells expandedWSGR Docket No.: 64421-706.601 with APL (SEQ ID NO: 12) showed increased IFNy spots as compared to the parental control (SEQ ID NO: 11), indicating superior T cell response (FIG. 4B).

[0188] Results shown in FIGS. 5A-FIG. 5B demonstrate that KRAS G12D APL1- HLA* 11 :01 binding and immunogenicity of engineered antigens show superior binding and T Cell responses. l * 106expanded CD8 T cells from no peptide, irrelevant peptide, parental peptide, or APL1 were seeded in an ELISpot capture plate coated with anti -human IFNy antibody. FIG. 5A shows non-linear regression curves and calculated EC50s for binding between HLA-A* 11 :01 and a parental KRAS G12D 10-mer peptide (SEQ ID NO: 11) and APL (SEQ ID NO: 77). 1 * 106expanded CD8 T cells treated with no peptide, irrelevant peptide, parental peptide, or APL were seeded in an ELISpot capture plate coated with anti-human IFNy antibody. T cells were stimulated with control or parental antigen overnight and IFNy capture was read out. Two-way ANOVA followed by uncorrected Fischer’s LSD test was performed. (ns = P > 0.05, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). CD8 T cells expanded with APL (SEQ ID NO: 77) showed increased IFNy spots as compared to the parental control (SEQ ID NO: 11), indicating superior T cell response (FIG. 5B).

[0189] Summary

[0190] It was demonstrated using altered peptide ligands (‘APLs’) in a pancreatic mouse model (Panc02) that APLs have distinct structural features measured by in silico modeling that enhance their immunogenicity in mice compared with the parental peptide (FIG. 3A-3F). These data support the use of in silico structural modeling to engineer antigens.

[0191] Heteroclitic peptides, for example 10-mers for TP53 Y220C, IDH1 R132H, CTNNB1 S45F, EGFR L858R, or JAK2 V617F which are been modeled for, for example, HLA 11*01, may show enhanced T cell response compared to their respective parental peptide.

[0192] EXAMPLE 2: VALIDATION AND METHODS OF TREATMENT USING HETEROCLITIC NEOEPITOPE VACCINES

[0193] Binding and immunogenicity assays are carried out for epitope and HLA combinations as disclosed in Example 1. Mutated versions of the immunogenic peptides disclosed herein are generated which comprise one or more amino acid substitutions relative to the respective parental version of each peptide as described herein. Mutated versions of the immunogenic peptides which display enhanced immunogenicity are utilized as neoantigens for the generation of heteroclitic neoantigen complexes. Certain neoantigens are determined toWSGR Docket No.: 64421-706.601 have enhanced immunogenicity relative to their respective parental peptides. Certain mutations are determined to generate neoantigens with enhanced immunogenicity relative to their respective parental peptides. Heteroclitic neoantigen complexes are generated for immunogenic peptides which display enhanced immunogenicity.

[0194] Heteroclitic neoantigen complexes with enhanced immunogenicity are used to generate neoantigen-targeted human vaccines. The neoantigen-targeted human vaccines are administered to human patients. The neoantigen-targeted human vaccines are used to treat one or more diseases such as cancer. The neoantigen-targeted human vaccines demonstrate efficacy in treating one or more cancers. The neoantigen-targeted human vaccines which demonstrate efficacy in treating one or more cancers are determined to have distinct structural features that enhance their immunogenicity. The neoantigen-targeted human vaccines which demonstrate efficacy in treating one or more cancers show enhanced T cell response compared to vaccines developed using their respective parental peptides. T cells demonstrating the enhanced response are characterized, and the T cell receptors which recognize the neoantigen-targeted human vaccines are further characterized.

[0195] EXAMPLE 3: KRAS HETEROCLITIC NEOEPITOPE VACCINES DERIVED FROM COMPUTATIONAL STRUCTURAL MODELING

[0196] A library of KRAS G12D, G12R, G12V, and G12C 10- and 9-mer altered peptide ligands (APLs) were generated using the corresponding parental epitopes based on the method as described in Example 1. Parental and APL sequences are shown in Table 2. Percent binding of KRAS G12D, G12R, G12V, and G12C 10- and 9-mer parental epitopes and altered peptide ligands is shown in FIG. 6. APLs with greater binding percentage relative to parental are shaded in gray.WSGR Docket No.: 64421-706.601

[0197] Table 2 Peptide sequences that correspond to APL and parental sequence (HLA- A *11:01)

[0198] Thus, these data support the use of in silico structural modeling to engineer antigens.OTHER EMBODIMENTS

[0199] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. It is understood that certain method steps may be reordered, removed or modified to perform the methods disclosed herein. Such embodiments are also within the scope of the following claims.

[0200] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

WSGR Docket No.: 64421-706.601CLAIMSWhat is claimed:

1. A method of identifying heteroclitic neoepitopes having improved immunogenicity, the method comprising:(a) modeling a library of peptides onto a human leukocyte antigen (HLA) to form an HLA-peptide structure, wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes;(b) selecting the HLA-peptide structure having a sequence identity similar to a parental epitope being modeled;(c) modeling heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position;(d) generating HLA-heteroclitic neoepitope complexes;(e) validating the HLA-heteroclitic neoepitope complexes using a root-mean-square deviation analysis (Ca-RMSD); and(f) performing a high-resolution Monte Carlo simulation with minimization docking of the heteroclitic neoepitope to the HLA cleft to identify the lowest full-atom energy conformation; thereby identifying the heteroclitic neoepitopes having improved immunogenicity.

2. The method of claim 1, wherein modeling the library of peptides onto the HLA comprises aligning peptide sequences with a consensus binding motif of HLA molecules.

3. The method of claim 2, wherein the consensus binding motif comprises a consensus binding motif for 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17- mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes.

4. The method of claim 3, wherein the consensus binding motif for the 8-mer, 9-mer, 10- mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21- mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprises distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors.

5. The method of any one of claims 1-4, wherein the root-mean-square deviation analysis is conducted to identify unstable structural changes of an HLA cleft.

6. The method of any one of claims 1-5, wherein anchor energetic-optimized structures are selected to identify anchor residue modifications.WSGR Docket No.: 64421-706.6017. The method of any one of claims 1-6, wherein the HLA-heteroclitic neoepitope complexes are generated to comprise combinations of anchor residue amino acids specific for each HLA class and subgroups thereof.

8. The method of any one of claims 1-7, wherein the HLA comprises an HLA type, wherein the HLA type comprises class I, II, or a subgroup thereof.

9. The method of any one of claims 1-8, wherein the peptides are derived from tumor cells or antigenic derivatives thereof.

10. The method of any one of claims 1-9, wherein the peptides comprise one or more tumor antigens.

11. The method of claim 10, wherein the one or more tumor antigens comprise one or more of tumor protein 53 (TP53), isocitrate dehydrogenase 1 (IDH1), catenin beta 1 (CTNNB1), epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2), Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma oncogene homolog Bl (BRAF), F-box and WD repeat domain containing 7 (FBWX7), fibroblast growth factor receptor 3 (FGFR3), mucin 4 (MUC4), neuroblastoma RAS viral oncogene homolog (NRAS), phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), protein phosphatase 2 regulatory subunit A alpha (PPP2R1 A), or phosphatase and tensin homolog (PTEN).

12. The method of claim 11, wherein the one or more tumor antigens comprise one or more mutations.

13. The method of claim 12, wherein the one or more mutations comprise TP53 Y220C.

14. The method of claim 12, wherein the one or more mutations comprise IDH1 R132H.

15. The method of claim 12, wherein the one or more mutations comprise CTNNB1 S45F.

16. The method of claim 12, wherein the one or more mutations comprise EGFR L858R.

17. The method of claim 12, wherein the one or more mutations comprise JAK2 V617F.

18. The method of any one of claims 1-17, wherein the HLA is HLA- A, HLA-B, or HLA- C.

19. The method of claim 18, wherein the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA- A*l l:01, HLA-A*30:01, or HLA-A*68:01.

20. The method of claim 18, wherein the HLA-B is HLA-B*07:02.WSGR Docket No.: 64421-706.60121. The method of claim 18, wherein the HLA-C is HLA-C*01 :02, HLA-C*03:03, HLA- C*03:04, or HLA-C*08:02.

22. The method of any one of claims 1-21, wherein the HLA-peptide structure shares a sequence identity of at least 85%, 90%, 95%, 97%, or 99% with the parental epitope.

23. A method of generating a library of immunogenic peptides, comprising: modeling a peptide onto a human leukocyte antigen (HLA), wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes; selecting an HLA-peptide structure having a sequence identity similar to a parental epitope being modeled; modeling a heteroclitic neoepitope into an HLA cleft by introducing amino acid rotamers with the lowest energy conformation at each position to form HLA-heteroclitic neoepitope complexes; validating the resulting HLA-heteroclitic neoepitope complexes using a root-meansquare deviation analysis (Ca-RMSD); and performing a high-resolution Monte Carlo simulation with minimization docking of the heteroclitic neoepitope to the HLA cleft to identify the lowest full-atom energy conformation; thereby generating the library of immunogenic peptides.

24. The method of claim 23, wherein modeling the heteroclitic neoepitope comprises introducing amino acid rotamers with a lowest energy conformation at each position.

25. The method of claim 23 or 24, wherein the root-mean-square deviation analysis is conducted to identify unstable structural changes of the HLA cleft.

26. The method of any one of claims 23-25, wherein the peptide epitopes comprise peptides isolated from tumor cells or antigenic derivatives thereof.

27. The method of any one of claims 23-26, wherein the peptide epitopes comprise one or more tumor antigens.

28. The method of claim 27, wherein the one or more tumor antigens comprise at least a portion of an amino acid sequence of one or more of TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN.

29. The method of claim 28, wherein the one or more tumor antigens comprises one or more mutations.

30. The method of claim 29, wherein the one or more mutations comprise TP53 Y220C.WSGR Docket No.: 64421-706.60131. The method of claim 29, wherein the one or more mutations comprise IDH1 R132H.

32. The method of claim 29, wherein the one or more mutations comprise CTNNB1 S45F.

33. The method of claim 29, wherein the one or more mutations comprise EGFR L858R.

34. The method of claim 29, wherein the one or more mutations comprise JAK2 V617F.

35. The method of any one of claims 27-34, wherein a sequence of a tumor antigen selected from the one or more tumor antigens is positionally aligned with 8-mer, 9-mer, 10-mer, 11- mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22- mer, 23-mer, 24-mer, or 25-mer epitopes that bind HLA molecules.

36. The method of claim 35, wherein the 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprise distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors.

37. The method of any one of claims 23-36, wherein a tumor antigen peptide comprising the greatest sequence similarity is selected for modeling into an HLA cleft.

38. The method of any one of claims 23-37, wherein anchor energetic-optimized HLA- heteroclitic neoepitope complexes are selected to identify anchor residue modifications.

39. The method of any one of claims 23-38, wherein a library of tumor antigen peptides is generated comprising combinations of anchor residue amino acids specific for each HLA class and subgroups thereof.

40. The method of claim 39, wherein the tumor antigen peptides comprise heteroclitic epitopes.

41. The method of claim 40, wherein contacting the tumor antigen peptides comprising heteroclitic epitopes with a population of immune cells leads to a higher percentage of expansion of the population of immune cells as compared to contacting with corresponding peptides that are otherwise the same but do not comprise the heteroclitic epitopes.

42. The method of claim 40, wherein contacting the tumor antigen peptides comprising heteroclitic epitopes with a population of immune cells leads to higher activation of the population of immune cells as compared to contacting with corresponding peptides that are otherwise the same but do not comprise the heteroclitic epitopes.

43. The method of claim 42, wherein the activation comprises IFNgamma secretion.WSGR Docket No.: 64421-706.60144. The method of any one of claims 23-43, wherein the HLA comprises an HLA type, wherein the HLA type comprises class I, class II, or a subgroup thereof.

45. The method of any one of claims 23-44, wherein the HLA is HLA-A, HLA-B, or HLA- C.

46. The method of claim 45, wherein the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA- A*l l:01, HLA-A*30:01, or HLA-A*68:01.

47. The method of claim 45, wherein the HLA-B is HLA-B*07:02.

48. The method of claim 45, wherein the HLA-C is HLA-C*01 :02, HLA-C*03:03, HLA- C*03:04, or HLA-C*08:02.

49. The method of any one of claims 1-48, further comprising quantifying differences in structural features between the peptide being modeled and the parental epitope.

50. The method of claim 49, wherein the structural features comprise contacts between the peptide and HLA cleft residues, solvent-accessible surface area (SASA), peptide rigidity as a measure of CaRMSD of the top 10 predicted structural models for each HLA-heteroclitic neoepitope complex, surface hydrophobicity, or electrostatic potential.

51. The method of claim 49 or 50, further comprising in vitro co-culturing of immunogenic peptides with HLA-matched lymphocytes to validate which immunogenic peptides induce the greatest T cell expansion and activation as compared to parental peptides, as assessed by IFN- gamma ELISpot assay.

52. A method of treating cancer comprising administering to a subject in need thereof one or more peptides comprising one or more heteroclitic epitopes.

53. The method of claim 52, wherein the one or more peptides are generated by the method of any one of claims 1-51.

54. The method of claim 52 or 53, further comprising administering an adjuvant to the subject.

55. The method of any one of claims 52-54, further comprising administering one or more therapeutics to the subject.

56. A cancer vaccine comprising one or more peptides, wherein the one or more peptides comprise at least one heteroclitic neoepitope.WSGR Docket No.: 64421-706.60157. The cancer vaccine of claim 56, wherein the one or more peptides comprise a tumor antigen.

58. The cancer vaccine of claim 57, wherein the tumor antigen comprises TP53, IDH1, CTNNB1, EGFR, JAK2, KRAS, BRAF, FBWX7, FGFR3, MUC4, NRAS, PIK3CA, PPP2R1 A, or PTEN59. The cancer vaccine of claim 58, wherein the tumor antigen comprises one or more mutations.

60. The cancer vaccine of claim 59, wherein the one or more mutations comprise TP53 Y220C.

61. The cancer vaccine of claim 59, wherein the one or more mutations comprise IDH1 R132H.

62. The cancer vaccine of claim 59, wherein the one or more mutations comprise CTNNB 1 S45F.

63. The cancer vaccine of claim 59, wherein the one or more mutations comprise EGFR L858R.

64. The cancer vaccine of claim 59, wherein the one or more mutations comprise JAK2 V617F.

65. The cancer vaccine of any one of claims 59-64, wherein the one or more peptides are displayed on a surface of an antigen presenting cell (APC) via an HLA antigen presentation pathway, and wherein a tumor specific immune response is induced.

66. The cancer vaccine of any one of claims 59-65, wherein the one or more peptides are immunogenic specifically for the subject’s HLA type.

67. The cancer vaccine of claim 66, wherein the HLA is HLA-A, HLA-B, or HLA-C.

68. The cancer vaccine of claim 66 or 67, wherein the HLA is HLA-A*01:01, HLA- A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*l l:01, HLA-A*23:01, HLA-A*30:01, HLA- A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B* 14:02, HLA-B* 18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51 :01,WSGR Docket No.: 64421-706.601HLA-B*53:01, HLA-B* 13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01 :02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C* 16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA- C*17:01.

69. The cancer vaccine of claim 67, wherein the HLA-A is HLA-A*02:01, HLA-A*03:01, HLA-A*l l:01, HLA-A*30:01, or HLA-A*68:01.

70. The cancer vaccine of claim 67, wherein the HLA-B is HLA-B*07:02.

71. The cancer vaccine of claim 67, wherein the HLA-C is HLA-C*01 :02, HLA-C*03:03,HLA-C*03:04, or HLA-C*08:02.

72. The cancer vaccine of any one of claims 56-71, wherein the at least one heteroclitic neoepitope is generated by the method of any one of claims 1-22 or wherein the one or more peptides comprise one or more immunogenic peptides generated by the method of any one of claims 23-51.

73. An expression vector encoding the one or more peptides of the cancer vaccine of any one of claims 56-72.

74. A method of treating cancer in a subject diagnosed with the cancer comprising: isolating cells from a biological sample of the subject; culturing the isolated cells with one or more peptide generated by the method of any one of claims 1-51, isolating T cells, NK cells, and / or antigen presenting cells cultured with the one or more peptide and expanding the T cells, NK cells, and / or antigen presenting cells to produce a therapeutically effective composition of tumor antigen specific T cells, NK cells, and / or antigen presenting cells; and adoptively transferring the tumor antigen specific T cells, NK cells, and / or antigen presenting cells into the subject, thereby treating the subject diagnosed with cancer.

75. The method of claim 74, wherein the one or more peptides comprise one or more heteroclitic neoepitopes.WSGR Docket No.: 64421-706.60176. A cell comprising one or more peptides generated by the method of any one of claims 1-51.

77. A pharmaceutical composition comprising one or more of a peptide generated by the method of any one of claims 1-51, the cancer vaccine of any one of claims 56-72, the cell of claim 76, the expression vector of claim 73, and a pharmaceutically acceptable excipient.

78. Use of one or more peptides generated by the method of any one of claims 1-51, the cancer vaccine of any one of claims 56-72, the expression vector of claim 73, the cell of claim 76, and / or the pharmaceutical composition of claim 77 in the manufacture of a medicament for treatment in a subject.

79. A kit comprising:(a) one or more peptides identified by the method of any one of claims 1-51, the cancer vaccine of any one of claims 56-72, the expression vector of claim 73, the cell of claim 76, the pharmaceutical composition of claim 77, and(b) an information material containing instructions for administering a dosage of the one or more peptides, the cancer vaccine, the cell, or a dosage form of the pharmaceutical composition to a subject.

79. A T cell receptor obtained via immunizing a subject with a peptide of any one of the previous claims and characterizing a subsequent immune response.

80. The method of any one of claims 52-54, 74, and 75, wherein the cancer is selected from the group consisting of: colorectal cancer, leukemia, Kaposi’s sarcoma, breast cancer, bone cancers, brain cancer, mantle cell lymphoma, non-Hodgkin’s lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, retinoblastoma, esophagus cancer, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer, pancreatic cancer, sarcomas, Wilms’ tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, and multidrug resistant cancer.

81. The method of claim 80, wherein the leukemia is selected from the group consisting of: acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia.WSGR Docket No.: 64421-706.60182. The method of claim 80, wherein the bone cancer is selected from the group consisting of: osteosarcoma, chondrosarcomas, Ewing’s sarcoma, fibrosarcomas, giant cell tumors, adamantinomas, and chordomas.

83. The method of claim 80, wherein the brain cancer is selected from the group consisting of: meningiomas, glioblastomas, lower-grade astrocytomas, oligodendrocytomas, pituitary tumor, schwannomas, primary CNS lymphoma, and metastatic brain cancers.

84. The method of claim 80, wherein the lung cancer is non-small cell lung carcinoma.

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