Compositions and methods for optimized peptide vaccines

Abstract

Described herein is a composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 184. Also described herein is a composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 222. Also described herein is a composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 223 to 386. Also described herein is a composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 387 to 794.

Description

Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 COMPOSITIONS AND METHODS FOR OPTIMIZED PEPTIDE VACCINES

[0001] This application claims the benefit of and priority under 35 U.S.C. § 119(e) to US Provisional Serial No.: 63 / 583,460, filed September 18, 2023, the contents of which is hereby incorporated by reference in its entirety.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights. INCORPORATION BY REFERENCE

[0003] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art. SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on September 9, 2024, is named 2215269_00133WO1_SL.xml and is 68,996,631 bytes in size. TECHNICAL FIELD

[0005] The present invention relates generally to compositions, systems, and methods of peptide vaccines. More particularly, the present invention relates to compositions, systems, and methods of designing peptide vaccines to treat or prevent disease optimized based on predicted population immunogenicity. BACKGROUND

[0006] The goal of a peptide vaccine is to train the immune system to recognize and expand its capacity to engage cells that display target peptides to improve the immune response to cancerous cells or pathogens. A peptide vaccine can also be administered to someone who is already diseased to increase their immune response to a causal cancer, other diseases, or pathogen. Alternatively, a peptide vaccine can be administered to induce the immune system to - 1 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 have therapeutic tolerance to one or more peptides. There exists a need for compositions, systems, and methods of peptide vaccines based on prediction of the target peptides that will be displayed to protect a host from cancer, other disease, or pathogen infection. SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides for a composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 17 to 176, and SEQ ID NOs: 178 to 184.

[0008] In some embodiments, the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides. In some embodiments, an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject. In some embodiments, the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele. In some embodiments, each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a CTNNB1 protein. In some embodiments, the fragment of the CTNNB1 protein comprises a mutation selected from the group consisting of CTNNB1 D32G, CTNNB1 G34E, CTNNB1 S33C, CTNNB1 S33F, CTNNB1 S37C, CTNNB1 S37F, CTNNB1 S45F, CTNNB1 S45P, CTNNB1 T41A, and CTNNB1 T41I.

[0009] In another aspect, the invention provides for a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the composition is an immunogenic composition. In some embodiments, the at least two amino acid sequences are SEQ ID NOs: 103 and 104.

[0010] In another aspect, the invention provides for composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 207 and SEQ ID NOs: 209 to 222. In some embodiments, the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least - 2 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 two peptides encoded by the one or more polynucleotides. In some embodiments, an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject. In some embodiments, the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele. In some embodiments, each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a KRAS protein. In some embodiments, the fragment of the KRAS protein comprises a mutation selected from the group consisting of KRAS A146T, KRAS A146V, and KRAS Q61H.

[0011] In another aspect, the invention provides for a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the composition is an immunogenic composition. In some embodiments, the at least two amino acid sequences are SEQ ID NOs: 203 and 204.

[0012] In another aspect, the invention provides for a composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 223 to 240, SEQ ID NOs: 243 to 293, SEQ ID NOs: 295 to 315, SEQ ID NOs: 317 to 329, SEQ ID NOs: 331 to 339, and SEQ ID NOs: 341 to 386. In some embodiments, the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides. In some embodiments, an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject. In some embodiments, the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele. In some embodiments, each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a PIK3CA protein. In some embodiments, the fragment of the PIK3CA protein comprises a mutation selected from the group consisting of PIK3CA C420R, - 3 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 PIK3CA E453K, PIK3CA E545A, PIK3CA E726K, PIK3CA G118D, PIK3CA H1047L, PIK3CA N345K, PIK3CA Q546K, PIK3CA Q546R, and PIK3CA R108H.

[0013] In another aspect, the invention provides for a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the composition is an immunogenic composition. In some embodiments, the at least two amino acid sequences are SEQ ID NOs: 308 and 309.

[0014] In another aspect, the invention provides for a composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 387 to 445, SEQ ID NOs: 447 to 472, SEQ ID NOs: 474 to 480, SEQ ID NOs: 482 to 500, SEQ ID NOs: 502 to 544, SEQ ID NOs: 546 to 550, SEQ ID NOs: 552 to 564, SEQ ID NOs: 566 to 582, SEQ ID NO: 584, SEQ ID NOs: 586 to 605, SEQ ID NOs: 607 to 633, SEQ ID NOs: 638 to 656, SEQ ID NOs: 659 to 664, and SEQ ID NOs: 666 to 794. In some embodiments, the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides. In some embodiments, an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject. In some embodiments, the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele. In some embodiments, each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a TP53 protein. In some embodiments, the fragment of the TP53 protein comprises a mutation selected from the group consisting of TP53 C176F, TP53 C176Y, TP53 C238Y, TP53 C275Y, TP53 E285K, TP53 G245S, TP53 G245V, TP53 H179Y, TP53 H193R, TP53 I195T, TP53 K132E, TP53 K132N, TP53 P151S, TP53 R213L, TP53 R249M, TP53 R249S, TP53 R273L, TP53 R280K, TP53 S127Y, TP53 S241F, TP53 V157F, TP53 V272M, TP53 Y163C, TP53 Y205C, and TP53 Y234C.

[0015] In another aspect, the invention provides for a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the compositions described herein. In some embodiments, the - 4 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 composition is an immunogenic composition. In some embodiments, the at least two amino acid sequences are SEQ ID NOs: 474 and 475.

[0016] In another aspect, the invention provides for nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834.

[0017] In some embodiments, the nucleic acid sequences encode two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834.

[0018] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834.

[0019] In some embodiments, the composition is administered to a subject. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834. In some embodiments, the nucleic acid sequences are contained in a construct for in vivo expression in a subject of at least one peptide encoded by the nucleic acid sequences. In some embodiments, an administration of the nucleic acid sequences causes the at least one peptide encoded by the nucleic acid sequences to be displayed by an HLA class I molecule in the subject. In some embodiments, the administration of the nucleic acid sequences causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated protein selected from the group consisting of BRAF, CTNNB1, KRAS, PIK3CA, and TP53. In some embodiments, the one or more peptides is a modified or unmodified fragment of a protein, wherein the protein comprises a mutation selected from the group consisting of BRAF G466V, CTNNB1 D32G, CTNNB1 G34E, CTNNB1 S33C, CTNNB1 S33F, CTNNB1 S37C, CTNNB1 S37F, CTNNB1 S45F, CTNNB1 S45P, CTNNB1 T41A, CTNNB1 T41I, KRAS A146T, KRAS A146V, KRAS Q61H, PIK3CA C420R, PIK3CA E453K, PIK3CA E545A, PIK3CA E726K, PIK3CA G118D, PIK3CA H1047L, PIK3CA N345K, PIK3CA Q546K, PIK3CA Q546R, PIK3CA R108H, TP53 C176F, TP53 C176Y, TP53 C238Y, TP53 C275Y, TP53 E285K, TP53 G245S, TP53 G245V, TP53 H179Y, TP53 H193R, TP53 I195T, TP53 K132E, TP53 K132N, TP53 P151S, TP53 R213L, TP53 R249M, TP53 R249S, TP53 R273L, TP53 R280K, TP53 S127Y, TP53 S241F, TP53 - 5 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 V157F, TP53 V272M, TP53 Y163C, TP53 Y205C, and TP53 Y234C. In some embodiments, the composition is administered in an effective amount to a subject to prevent cancer. In some embodiments, the composition is administered in an effective amount to a subject to treat cancer. In some embodiments, the cancer is selected from the group consisting of pancreas, colon, rectum, kidney, bronchus, lung, uterus, cervix, bladder, liver, stomach, brain, breast, ovary, thyroid, and skin. In some embodiments, the composition comprises nucleic acid sequences encoding at least three amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834.

[0020] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 834.

[0021] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1 to 834.

[0022] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0023] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a BRAF protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0024] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 16.

[0025] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1 to 16. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated BRAF protein.

[0026] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 184.

[0027] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 protein mutation. In some - 6 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 184.

[0028] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 17 to 184.

[0029] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 17 to 184. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated CTNNB1 protein.

[0030] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 222.

[0031] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 222.

[0032] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 185 to 222.

[0033] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 185 to 222. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated KRAS protein.

[0034] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 223 to 386.

[0035] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 223 to 386.

[0036] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 223 to 386. - 7 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0037] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 223 to 386. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated PIK3CA protein.

[0038] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 387 to 794.

[0039] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 387 to 794.

[0040] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 387 to 794.

[0041] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 387 to 794. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated TP53 protein.

[0042] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0043] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a BRAF G466V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16.

[0044] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 16.

[0045] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1 to 16. In some embodiments, the peptide composition comprises a peptide derived from a BRAF G466V protein mutation.

[0046] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 36. - 8 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0047] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 D32G protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 36.

[0048] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 17 to 36.

[0049] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 17 to 36. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 D32G protein mutation.

[0050] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 37 to 52.

[0051] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 G34E protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 37 to 52.

[0052] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 37 to 52.

[0053] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 37 to 52. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 G34E protein mutation.

[0054] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 53 to 67.

[0055] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S33C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 53 to 67.

[0056] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 53 to 67. - 9 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0057] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 53 to 67. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S33C protein mutation.

[0058] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68 to 86.

[0059] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S33F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68 to 86.

[0060] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 68 to 86.

[0061] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 68 to 86. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S33F protein mutation.

[0062] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 87 to 102.

[0063] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S37C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 87 to 102.

[0064] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 87 to 102.

[0065] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 87 to 102. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S37C protein mutation.

[0066] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 103 to 119. - 10 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0067] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S37F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 103 to 119.

[0068] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 103 to 119.

[0069] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 103 to 119. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S37F protein mutation.

[0070] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 120 to 134.

[0071] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S45F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 120 to 134.

[0072] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 120 to 134.

[0073] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 120 to 134. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S45F protein mutation.

[0074] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 135 to 150.

[0075] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S45P protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 135 to 150.

[0076] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 135 to 150. - 11 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0077] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 135 to 150. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S45P protein mutation.

[0078] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 151 to 167.

[0079] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 T41A protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 151 to 167.

[0080] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 151 to 167.

[0081] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 151 to 167. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 T41A protein mutation.

[0082] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 168 to 184.

[0083] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 T41I protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 168 to 184.

[0084] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 168 to 184.

[0085] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 168 to 184. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 T41I protein mutation.

[0086] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 193. - 12 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0087] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS A146T protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 193.

[0088] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 185 to 193.

[0089] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 185 to 193. In some embodiments, the peptide composition comprises a peptide derived from a KRAS A146T protein mutation.

[0090] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 194 to 202.

[0091] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS A146V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 194 to 202.

[0092] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 194 to 202.

[0093] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 194 to 202. In some embodiments, the peptide composition comprises a peptide derived from a KRAS A146V protein mutation.

[0094] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 203 to 222.

[0095] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS Q61H protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 203 to 222.

[0096] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 203 to 222. - 13 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0097] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 203 to 222. In some embodiments, the peptide composition comprises a peptide derived from a KRAS Q61H protein mutation.

[0098] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 223 to 239.

[0099] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA C420R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 223 to 239.

[0100] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 223 to 239.

[0101] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 223 to 239. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA C420R protein mutation.

[0102] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 240 to 255.

[0103] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E453K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 240 to 255.

[0104] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 240 to 255.

[0105] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 240 to 255. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E453K protein mutation.

[0106] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 256 to 271. - 14 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0107] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E545A protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 256 to 271.

[0108] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 256 to 271.

[0109] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 256 to 271. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E545A protein mutation.

[0110] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 272 to 290.

[0111] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E726K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 272 to 290.

[0112] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 272 to 290.

[0113] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 272 to 290. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E726K protein mutation.

[0114] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 291 to 307.

[0115] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA G118D protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 291 to 307.

[0116] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 291 to 307. - 15 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0117] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 291 to 307. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA G118D protein mutation.

[0118] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 308 to 325.

[0119] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA H1047L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 308 to 325.

[0120] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 308 to 325.

[0121] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 308 to 325. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA H1047L protein mutation.

[0122] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 326 to 339.

[0123] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA N345K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 326 to 339.

[0124] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 326 to 339.

[0125] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 326 to 339. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA N345K protein mutation.

[0126] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 340 to 358. - 16 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0127] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA Q546K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 340 to 358.

[0128] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 340 to 358.

[0129] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 340 to 358. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA Q546K protein mutation.

[0130] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 359 to 376.

[0131] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA Q546R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 359 to 376.

[0132] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 359 to 376.

[0133] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 359 to 376. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA Q546R protein mutation.

[0134] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 377 to 386.

[0135] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA R108H protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 377 to 386.

[0136] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 377 to 386. - 17 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0137] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 377 to 386. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA R108H protein mutation.

[0138] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 387 to 403.

[0139] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C176F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 387 to 403.

[0140] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 387 to 403.

[0141] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 387 to 403. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C176F protein mutation.

[0142] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 404 to 420.

[0143] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C176Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 404 to 420.

[0144] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 404 to 420.

[0145] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 404 to 420. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C176Y protein mutation.

[0146] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 421 to 440. - 18 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0147] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C238Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 421 to 440.

[0148] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 421 to 440.

[0149] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 421 to 440. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C238Y protein mutation.

[0150] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 441 to 457.

[0151] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C275Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 441 to 457.

[0152] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 441 to 457.

[0153] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 441 to 457. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C275Y protein mutation.

[0154] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 458 to 473.

[0155] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 E285K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 458 to 473.

[0156] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 458 to 473. - 19 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0157] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 458 to 473. In some embodiments, the peptide composition comprises a peptide derived from a TP53 E285K protein mutation.

[0158] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 474 to 492.

[0159] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 G245S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 474 to 492.

[0160] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 474 to 492.

[0161] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 474 to 492. In some embodiments, the peptide composition comprises a peptide derived from a TP53 G245S protein mutation.

[0162] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 493 to 511.

[0163] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 G245V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 493 to 511.

[0164] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 493 to 511.

[0165] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 493 to 511. In some embodiments, the peptide composition comprises a peptide derived from a TP53 G245V protein mutation.

[0166] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 512 to 529. - 20 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0167] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 H179Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 512 to 529.

[0168] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 512 to 529.

[0169] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 512 to 529. In some embodiments, the peptide composition comprises a peptide derived from a TP53 H179Y protein mutation.

[0170] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 530 to 546.

[0171] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 H193R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 530 to 546.

[0172] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 530 to 546.

[0173] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 530 to 546. In some embodiments, the peptide composition comprises a peptide derived from a TP53 H193R protein mutation.

[0174] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 547 to 563.

[0175] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 I195T protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 547 to 563.

[0176] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 547 to 563. - 21 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0177] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 547 to 563. In some embodiments, the peptide composition comprises a peptide derived from a TP53 I195T protein mutation.

[0178] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 564 to 582.

[0179] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 K132E protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 564 to 582.

[0180] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 564 to 582.

[0181] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 564 to 582. In some embodiments, the peptide composition comprises a peptide derived from a TP53 K132E protein mutation.

[0182] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 583 to 599.

[0183] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 K132N protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 583 to 599.

[0184] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 583 to 599.

[0185] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 583 to 599. In some embodiments, the peptide composition comprises a peptide derived from a TP53 K132N protein mutation.

[0186] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 600 to 614. - 22 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0187] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 P151S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 600 to 614.

[0188] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 600 to 614.

[0189] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 600 to 614. In some embodiments, the peptide composition comprises a peptide derived from a TP53 P151S protein mutation.

[0190] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 615 to 631.

[0191] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R213L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 615 to 631.

[0192] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 615 to 631.

[0193] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 615 to 631. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R213L protein mutation.

[0194] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 632 to 648.

[0195] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R249M protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 632 to 648.

[0196] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 632 to 648. - 23 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0197] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 632 to 648. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R249M protein mutation.

[0198] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 649 to 665.

[0199] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R249S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 649 to 665.

[0200] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 649 to 665.

[0201] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 649 to 665. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R249S protein mutation.

[0202] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 666 to 680.

[0203] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R273L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 666 to 680.

[0204] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 666 to 680.

[0205] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 666 to 680. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R273L protein mutation.

[0206] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NO: 681. - 24 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0207] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R280K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NO: 681.

[0208] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NO: 681.

[0209] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NO: 681. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R280K protein mutation.

[0210] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 682 to 700.

[0211] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 S127Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 682 to 700.

[0212] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 682 to 700.

[0213] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 682 to 700. In some embodiments, the peptide composition comprises a peptide derived from a TP53 S127Y protein mutation.

[0214] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 701 to 718.

[0215] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 S241F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 701 to 718.

[0216] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 701 to 718. - 25 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0217] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 701 to 718. In some embodiments, the peptide composition comprises a peptide derived from a TP53 S241F protein mutation.

[0218] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 719 to 738.

[0219] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 V157F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 719 to 738.

[0220] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 719 to 738.

[0221] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 719 to 738. In some embodiments, the peptide composition comprises a peptide derived from a TP53 V157F protein mutation.

[0222] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 739 to 755.

[0223] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 V272M protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 739 to 755.

[0224] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 739 to 755.

[0225] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 739 to 755. In some embodiments, the peptide composition comprises a peptide derived from a TP53 V272M protein mutation.

[0226] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 756 to 772. - 26 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0227] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 Y163C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 756 to 772.

[0228] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 756 to 772.

[0229] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 756 to 772. In some embodiments, the peptide composition comprises a peptide derived from a TP53 Y163C protein mutation.

[0230] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 773 to 783.

[0231] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 Y205C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 773 to 783.

[0232] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 773 to 783.

[0233] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 773 to 783. In some embodiments, the peptide composition comprises a peptide derived from a TP53 Y205C protein mutation.

[0234] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 784 to 794.

[0235] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 Y234C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 784 to 794.

[0236] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 784 to 794. - 27 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0237] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 784 to 794. In some embodiments, the peptide composition comprises a peptide derived from a TP53 Y234C protein mutation.

[0238] In one aspect, the invention provides for nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0239] In some embodiments, the nucleic acid sequences encode two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0240] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0241] In some embodiments, the composition is administered to a subject. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279. In some embodiments, the nucleic acid sequences are contained in a construct for in vivo expression in a subject of at least one peptide encoded by the nucleic acid sequences. In some embodiments, an administration of the nucleic acid sequences causes the at least one peptide encoded by the nucleic acid sequences to be displayed by an HLA class II molecule in the subject. In some embodiments, the administration of the nucleic acid sequences causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class II alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class II alleles, wherein the first plurality of HLA class II alleles and the second plurality of HLA class II alleles differ by at least one HLA class II allele. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated protein selected from the group consisting of BRAF, CTNNB1, KRAS, PIK3CA, and TP53. In some embodiments, the one or more peptides is a modified or unmodified fragment of a protein, wherein the protein comprises a mutation selected from the group consisting of BRAF G466V, CTNNB1 D32G, CTNNB1 G34E, CTNNB1 S33C, CTNNB1 S33F, CTNNB1 S37C, CTNNB1 S37F, CTNNB1 S45F, CTNNB1 S45P, CTNNB1 T41A, CTNNB1 T41I, KRAS A146T, KRAS A146V, KRAS Q61H, PIK3CA C420R, PIK3CA E453K, PIK3CA E545A, PIK3CA E726K, PIK3CA G118D, PIK3CA H1047L, PIK3CA N345K, PIK3CA Q546K, PIK3CA Q546R, PIK3CA R108H, TP53 C176F, TP53 C176Y, TP53 C238Y, TP53 C275Y, TP53 G245S, TP53 G245V, TP53 H179Y, TP53 H193R, TP53 I195T, TP53 K132E, TP53 K132N, TP53 P151S, TP53 R213L, TP53 R249M, - 28 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 TP53 R249S, TP53 R273L, TP53 S127Y, TP53 S241F, TP53 V157F, TP53 V272M, TP53 Y163C, and TP53 Y205C. In some embodiments, the composition is administered in an effective amount to a subject to prevent cancer. In some embodiments, the composition is administered in an effective amount to a subject to treat cancer. In some embodiments, the cancer is selected from the group consisting of pancreas, colon, rectum, kidney, bronchus, lung, uterus, cervix, bladder, liver, stomach, brain, breast, ovary, thyroid, and skin. In some embodiments, the composition comprises nucleic acid sequences encoding at least three amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0242] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0243] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 835 to 1279.

[0244] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 844.

[0245] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a BRAF protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 844.

[0246] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 835 to 844.

[0247] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 835 to 844. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated BRAF protein.

[0248] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 845 to 935.

[0249] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 protein mutation. In some - 29 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 845 to 935.

[0250] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 845 to 935.

[0251] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 845 to 935. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated CTNNB1 protein.

[0252] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 936 to 965.

[0253] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 936 to 965.

[0254] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 936 to 965.

[0255] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 936 to 965. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated KRAS protein.

[0256] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 966 to 1065.

[0257] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 966 to 1065.

[0258] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 966 to 1065. - 30 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0259] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 966 to 1065. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated PIK3CA protein.

[0260] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1066 to 1279.

[0261] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1066 to 1279.

[0262] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1066 to 1279.

[0263] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1066 to 1279. In some embodiments, the one or more peptides is a modified or unmodified fragment of a mutated TP53 protein.

[0264] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 844.

[0265] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a BRAF G466V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 844.

[0266] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 835 to 844.

[0267] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 835 to 844. In some embodiments, the peptide composition comprises a peptide derived from a BRAF G466V protein mutation.

[0268] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 845 to 854. - 31 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0269] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 D32G protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 845 to 854.

[0270] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 845 to 854.

[0271] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 845 to 854. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 D32G protein mutation.

[0272] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 855 to 864.

[0273] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 G34E protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 855 to 864.

[0274] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 855 to 864.

[0275] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 855 to 864. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 G34E protein mutation.

[0276] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NO: 865.

[0277] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S33C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NO: 865.

[0278] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NO: 865. - 32 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0279] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NO: 865. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S33C protein mutation.

[0280] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 866 to 875.

[0281] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S33F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 866 to 875.

[0282] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 866 to 875.

[0283] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 866 to 875. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S33F protein mutation.

[0284] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 876 to 885.

[0285] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S37C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 876 to 885.

[0286] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 876 to 885.

[0287] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 876 to 885. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S37C protein mutation.

[0288] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 886 to 895. - 33 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0289] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S37F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 886 to 895.

[0290] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 886 to 895.

[0291] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 886 to 895. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S37F protein mutation.

[0292] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 896 to 905.

[0293] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S45F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 896 to 905.

[0294] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 896 to 905.

[0295] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 896 to 905. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S45F protein mutation.

[0296] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 906 to 915.

[0297] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 S45P protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 906 to 915.

[0298] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 906 to 915. - 34 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0299] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 906 to 915. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 S45P protein mutation.

[0300] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 916 to 925.

[0301] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 T41A protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 916 to 925.

[0302] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 916 to 925.

[0303] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 916 to 925. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 T41A protein mutation.

[0304] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 926 to 935.

[0305] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a CTNNB1 T41I protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 926 to 935.

[0306] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 926 to 935.

[0307] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 926 to 935. In some embodiments, the peptide composition comprises a peptide derived from a CTNNB1 T41I protein mutation.

[0308] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 936 to 945. - 35 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0309] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS A146T protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 936 to 945.

[0310] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 936 to 945.

[0311] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 936 to 945. In some embodiments, the peptide composition comprises a peptide derived from a KRAS A146T protein mutation.

[0312] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 946 to 955.

[0313] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS A146V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 946 to 955.

[0314] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 946 to 955.

[0315] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 946 to 955. In some embodiments, the peptide composition comprises a peptide derived from a KRAS A146V protein mutation.

[0316] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 956 to 965.

[0317] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a KRAS Q61H protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 956 to 965.

[0318] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 956 to 965. - 36 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0319] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 956 to 965. In some embodiments, the peptide composition comprises a peptide derived from a KRAS Q61H protein mutation.

[0320] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 966 to 975.

[0321] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA C420R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 966 to 975.

[0322] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 966 to 975.

[0323] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 966 to 975. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA C420R protein mutation.

[0324] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 976 to 985.

[0325] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E453K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 976 to 985.

[0326] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 976 to 985.

[0327] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 976 to 985. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E453K protein mutation.

[0328] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 986 to 995. - 37 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0329] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E545A protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 986 to 995.

[0330] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 986 to 995.

[0331] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 986 to 995. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E545A protein mutation.

[0332] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 996 to 1005.

[0333] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA E726K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 996 to 1005.

[0334] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 996 to 1005.

[0335] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 996 to 1005. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA E726K protein mutation.

[0336] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1006 to 1015.

[0337] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA G118D protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1006 to 1015.

[0338] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1006 to 1015. - 38 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0339] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1006 to 1015. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA G118D protein mutation.

[0340] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1016 to 1025.

[0341] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA H1047L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1016 to 1025.

[0342] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1016 to 1025.

[0343] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1016 to 1025. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA H1047L protein mutation.

[0344] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1026 to 1035.

[0345] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA N345K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1026 to 1035.

[0346] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1026 to 1035.

[0347] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1026 to 1035. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA N345K protein mutation.

[0348] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1036 to 1045. - 39 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0349] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA Q546K protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1036 to 1045.

[0350] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1036 to 1045.

[0351] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1036 to 1045. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA Q546K protein mutation.

[0352] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1046 to 1055.

[0353] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA Q546R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1046 to 1055.

[0354] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1046 to 1055.

[0355] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1046 to 1055. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA Q546R protein mutation.

[0356] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1056 to 1065.

[0357] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a PIK3CA R108H protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1056 to 1065.

[0358] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1056 to 1065. - 40 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0359] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1056 to 1065. In some embodiments, the peptide composition comprises a peptide derived from a PIK3CA R108H protein mutation.

[0360] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1066 to 1075.

[0361] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C176F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1066 to 1075.

[0362] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1066 to 1075.

[0363] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1066 to 1075. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C176F protein mutation.

[0364] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1076 to 1085.

[0365] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C176Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1076 to 1085.

[0366] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1076 to 1085.

[0367] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1076 to 1085. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C176Y protein mutation.

[0368] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1086 to 1095. - 41 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0369] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C238Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1086 to 1095.

[0370] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1086 to 1095.

[0371] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1086 to 1095. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C238Y protein mutation.

[0372] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1096 to 1105.

[0373] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 C275Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1096 to 1105.

[0374] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1096 to 1105.

[0375] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1096 to 1105. In some embodiments, the peptide composition comprises a peptide derived from a TP53 C275Y protein mutation.

[0376] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1106 to 1115.

[0377] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 G245S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1106 to 1115.

[0378] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1106 to 1115. - 42 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0379] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1106 to 1115. In some embodiments, the peptide composition comprises a peptide derived from a TP53 G245S protein mutation.

[0380] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1116 to 1125.

[0381] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 G245V protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1116 to 1125.

[0382] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1116 to 1125.

[0383] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1116 to 1125. In some embodiments, the peptide composition comprises a peptide derived from a TP53 G245V protein mutation.

[0384] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1126 to 1135.

[0385] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 H179Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1126 to 1135.

[0386] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1126 to 1135.

[0387] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1126 to 1135. In some embodiments, the peptide composition comprises a peptide derived from a TP53 H179Y protein mutation.

[0388] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1136 to 1145. - 43 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0389] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 H193R protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1136 to 1145.

[0390] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1136 to 1145.

[0391] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1136 to 1145. In some embodiments, the peptide composition comprises a peptide derived from a TP53 H193R protein mutation.

[0392] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1146 to 1155.

[0393] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 I195T protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1146 to 1155.

[0394] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1146 to 1155.

[0395] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1146 to 1155. In some embodiments, the peptide composition comprises a peptide derived from a TP53 I195T protein mutation.

[0396] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1156 to 1165.

[0397] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 K132E protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1156 to 1165.

[0398] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1156 to 1165. - 44 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0399] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1156 to 1165. In some embodiments, the peptide composition comprises a peptide derived from a TP53 K132E protein mutation.

[0400] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1166 to 1175.

[0401] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 K132N protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1166 to 1175.

[0402] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1166 to 1175.

[0403] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1166 to 1175. In some embodiments, the peptide composition comprises a peptide derived from a TP53 K132N protein mutation.

[0404] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1176 to 1185.

[0405] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 P151S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1176 to 1185.

[0406] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1176 to 1185.

[0407] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1176 to 1185. In some embodiments, the peptide composition comprises a peptide derived from a TP53 P151S protein mutation.

[0408] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1186 to 1195. - 45 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0409] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R213L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1186 to 1195.

[0410] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1186 to 1195.

[0411] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1186 to 1195. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R213L protein mutation.

[0412] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1196 to 1205.

[0413] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R249M protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1196 to 1205.

[0414] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1196 to 1205.

[0415] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1196 to 1205. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R249M protein mutation.

[0416] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1206 to 1215.

[0417] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R249S protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1206 to 1215.

[0418] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1206 to 1215. - 46 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0419] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1206 to 1215. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R249S protein mutation.

[0420] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1216 to 1225.

[0421] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 R273L protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1216 to 1225.

[0422] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1216 to 1225.

[0423] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1216 to 1225. In some embodiments, the peptide composition comprises a peptide derived from a TP53 R273L protein mutation.

[0424] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1226 to 1235.

[0425] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 S127Y protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1226 to 1235.

[0426] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1226 to 1235.

[0427] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1226 to 1235. In some embodiments, the peptide composition comprises a peptide derived from a TP53 S127Y protein mutation.

[0428] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1236 to 1245. - 47 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0429] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 S241F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1236 to 1245.

[0430] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1236 to 1245.

[0431] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1236 to 1245. In some embodiments, the peptide composition comprises a peptide derived from a TP53 S241F protein mutation.

[0432] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1246 to 1255.

[0433] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 V157F protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1246 to 1255.

[0434] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1246 to 1255.

[0435] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1246 to 1255. In some embodiments, the peptide composition comprises a peptide derived from a TP53 V157F protein mutation.

[0436] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1256 to 1259.

[0437] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 V272M protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1256 to 1259.

[0438] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1256 to 1259. - 48 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0439] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1256 to 1259. In some embodiments, the peptide composition comprises a peptide derived from a TP53 V272M protein mutation.

[0440] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1260 to 1269.

[0441] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 Y163C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1260 to 1269.

[0442] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1260 to 1269.

[0443] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1260 to 1269. In some embodiments, the peptide composition comprises a peptide derived from a TP53 Y163C protein mutation.

[0444] In another aspect, the invention provides for a composition comprising nucleic acid sequences encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1270 to 1279.

[0445] In some embodiments, the composition comprises nucleic acid sequences encoding one or more amino acid sequences derived from a TP53 Y205C protein mutation. In some embodiments, the composition comprises nucleic acid sequences encoding two or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1270 to 1279.

[0446] In another aspect, the invention provides for a peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1270 to 1279.

[0447] In some embodiments, the peptide composition comprises two or more peptides selected from the group consisting of SEQ ID NOs: 1270 to 1279. In some embodiments, the peptide composition comprises a peptide derived from a TP53 Y205C protein mutation.

[0448] In some embodiments, the compositions, including peptide compositions, of the invention are immunogenic compositions. To this end, the invention provides for a method of - 49 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 inducing an immunogenic response in a subject comprising administering to the subject a composition of the invention.

[0449] In some embodiments, compositions, including peptide compositions, of the invention are vaccines.

[0450] In one aspect, a composition (e.g., a vaccine) comprises nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834 and SEQ ID NOs: 1280 to 36796, wherein the one or more amino acid sequences encodes for one of more peptides, wherein the one or more peptides is configured to bind in vivo to one or more HLA alleles present in a subject, and wherein the one or more HLA alleles present in the subject is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, and C1803.

[0451] In one aspect, a method is described comprising administering to a subject a composition (e.g., a vaccine) in an effective amount to induce an immune response in the - 50 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 subject, the composition comprising nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834 and SEQ ID NOs: 1280 to 36796.

[0452] In some embodiments, the method comprises administering the composition to the subject based on a determination that the subject expresses one or more HLA alleles. In some embodiments, the one or more HLA alleles is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, and C1803.

[0453] In one aspect, a method is described for formulating a composition (e.g., a vaccine), the method comprising determining that a subject in need thereof of the composition expresses one or more HLA alleles, and formulating the composition by selecting nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 834 and SEQ ID NOs: 1280 to 36796. - 51 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0454] In some embodiments, the one or more HLA alleles is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, and C1803.

[0455] In another aspect, an MHC class II peptide vaccine comprises nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279 and SEQ ID NOs: 36797 to 61281, wherein the one or more amino acid sequences encodes for one of more peptides, wherein the one or more peptides is configured to bind in vivo to one or more HLA alleles present in a subject, and wherein the one or more HLA alleles present in the subject is selected from the group consisting of DPA10103-DPB10101, DPA10103-DPB10201, DPA10103-DPB10202, DPA10103-DPB10301, DPA10103- DPB10401, DPA10103-DPB10402, DPA10103-DPB10501, DPA10103-DPB10601, DPA10103-DPB11001, DPA10103-DPB110501, DPA10103-DPB11101, DPA10103- DPB112401, DPA10103-DPB112601, DPA10103-DPB11301, DPA10103-DPB113101, DPA10103-DPB113301, DPA10103-DPB11401, DPA10103-DPB11501, DPA10103- DPB11601, DPA10103-DPB11701, DPA10103-DPB11801, DPA10103-DPB11901, - 52 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPA10103-DPB12001, DPA10103-DPB12101, DPA10103-DPB12301, DPA10103- DPB12901, DPA10103-DPB13001, DPA10103-DPB13101, DPA10103-DPB13401, DPA10103-DPB13901, DPA10103-DPB14001, DPA10103-DPB14101, DPA10103- DPB14801, DPA10103-DPB14901, DPA10103-DPB15001, DPA10103-DPB15101, DPA10103-DPB15501, DPA10103-DPB15901, DPA10103-DPB18001, DPA10104- DPB10101, DPA10104-DPB10301, DPA10104-DPB11001, DPA10104-DPB11301, DPA10104-DPB11501, DPA10105-DPB10101, DPA10105-DPB10301, DPA10105- DPB10401, DPA10105-DPB11801, DPA10105-DPB15001, DPA10106-DPB10101, DPA10106-DPB11101, DPA10201-DPB10101, DPA10201-DPB10201, DPA10201- DPB10202, DPA10201-DPB10301, DPA10201-DPB10401, DPA10201-DPB10501, DPA10201-DPB10601, DPA10201-DPB110601, DPA10201-DPB11101, DPA10201- DPB11301, DPA10201-DPB113301, DPA10201-DPB11401, DPA10201-DPB11501, DPA10201-DPB11601, DPA10201-DPB11801, DPA10201-DPB11901, DPA10201- DPB13401, DPA10201-DPB14501, DPA10201-DPB15501, DPA10201-DPB19101, DPA10202-DPB10101, DPA10202-DPB10201, DPA10202-DPB10202, DPA10202- DPB10301, DPA10202-DPB10401, DPA10202-DPB10402, DPA10202-DPB10501, DPA10202-DPB10901, DPA10202-DPB110001, DPA10202-DPB110501, DPA10202- DPB110601, DPA10202-DPB11101, DPA10202-DPB11301, DPA10202-DPB113101, DPA10202-DPB11401, DPA10202-DPB11701, DPA10202-DPB11801, DPA10202- DPB11901, DPA10202-DPB12101, DPA10202-DPB12901, DPA10202-DPB13001, DPA10202-DPB13101, DPA10202-DPB13801, DPA10202-DPB14501, DPA10202- DPB15501, DPA10202-DPB16501, DPA10202-DPB18501, DPA10301-DPB10101, DPA10301-DPB10201, DPA10301-DPB10202, DPA10301-DPB10301, DPA10301- DPB10401, DPA10301-DPB10402, DPA10301-DPB10901, DPA10301-DPB110501, DPA10301-DPB110601, DPA10301-DPB11101, DPA10301-DPB11301, DPA10301- DPB11701, DPA10301-DPB11801, DPA10301-DPB12301, DPA10301-DPB13901, DPA10301-DPB14001, DPA10301-DPB14901, DPA10301-DPB15501, DPA10301- DPB16501, DPA10301-DPB17501, DPA10301-DPB18001, DPA10401-DPB10101, DPA10401-DPB10201, DPA10401-DPB10202, DPA10401-DPB10301, DPA10401- DPB10402, DPA10401-DPB10501, DPA10401-DPB110501, DPA10401-DPB11301, DPA10401-DPB113301, DPA10401-DPB11401, DPA10401-DPB14901, DQA10101- DQB10501, DQA10101-DQB10601, DQA10102-DQB10501, DQA10102-DQB10502, DQA10102-DQB10503, DQA10102-DQB10601, DQA10102-DQB10602, DQA10102- - 53 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DQB10604, DQA10102-DQB10609, DQA10102-DQB10611, DQA10103-DQB10501, DQA10103-DQB10503, DQA10103-DQB10601, DQA10103-DQB10602, DQA10103- DQB10609, DQA10104-DQB10501, DQA10105-DQB10501, DQA10201-DQB10301, DQA10201-DQB10302, DQA10201-DQB10303, DQA10301-DQB10301, DQA10301- DQB10302, DQA10301-DQB10303, DQA10301-DQB10304, DQA10302-DQB10301, DQA10302-DQB10302, DQA10302-DQB10303, DQA10302-DQB10304, DQA10303- DQB10301, DQA10303-DQB10302, DQA10303-DQB10303, DQA10303-DQB10304, DQA10401-DQB10301, DQA10401-DQB10319, DQA10501-DQB10201, DQA10501- DQB10203, DQA10501-DQB10301, DQA10501-DQB10319, DQA10501-DQB10501, DQA10503-DQB10301, DQA10505-DQB10201, DQA10505-DQB10202, DQA10505- DQB10301, DQA10505-DQB10302, DQA10505-DQB10309, DQA10505-DQB10319, DQA10505-DQB10402, DQA10505-DQB10501, DQA10506-DQB10303, DQA10508- DQB10301, DQA10509-DQB10301, DQA10601-DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602.

[0456] In one aspect, a method is described comprising administering to a subject a composition (e.g., a vaccine) in an effective amount to induce an immune response in the subject, the composition comprising nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279 and SEQ ID NOs: 36797 to 61281.

[0457] In some embodiments, the method comprises administering the composition to the subject based on a determination that the subject expresses one or more HLA alleles. In some embodiments, the one or more HLA alleles is selected from the group consisting of DPA10103- DPB10101, DPA10103-DPB10201, DPA10103-DPB10202, DPA10103-DPB10301, DPA10103-DPB10401, DPA10103-DPB10402, DPA10103-DPB10501, DPA10103- DPB10601, DPA10103-DPB11001, DPA10103-DPB110501, DPA10103-DPB11101, DPA10103-DPB112401, DPA10103-DPB112601, DPA10103-DPB11301, DPA10103- - 54 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPB113101, DPA10103-DPB113301, DPA10103-DPB11401, DPA10103-DPB11501, DPA10103-DPB11601, DPA10103-DPB11701, DPA10103-DPB11801, DPA10103- DPB11901, DPA10103-DPB12001, DPA10103-DPB12101, DPA10103-DPB12301, DPA10103-DPB12901, DPA10103-DPB13001, DPA10103-DPB13101, DPA10103- DPB13401, DPA10103-DPB13901, DPA10103-DPB14001, DPA10103-DPB14101, DPA10103-DPB14801, DPA10103-DPB14901, DPA10103-DPB15001, DPA10103- DPB15101, DPA10103-DPB15501, DPA10103-DPB15901, DPA10103-DPB18001, DPA10104-DPB10101, DPA10104-DPB10301, DPA10104-DPB11001, DPA10104- DPB11301, DPA10104-DPB11501, DPA10105-DPB10101, DPA10105-DPB10301, DPA10105-DPB10401, DPA10105-DPB11801, DPA10105-DPB15001, DPA10106- DPB10101, DPA10106-DPB11101, DPA10201-DPB10101, DPA10201-DPB10201, DPA10201-DPB10202, DPA10201-DPB10301, DPA10201-DPB10401, DPA10201- DPB10501, DPA10201-DPB10601, DPA10201-DPB110601, DPA10201-DPB11101, DPA10201-DPB11301, DPA10201-DPB113301, DPA10201-DPB11401, DPA10201- DPB11501, DPA10201-DPB11601, DPA10201-DPB11801, DPA10201-DPB11901, DPA10201-DPB13401, DPA10201-DPB14501, DPA10201-DPB15501, DPA10201- DPB19101, DPA10202-DPB10101, DPA10202-DPB10201, DPA10202-DPB10202, DPA10202-DPB10301, DPA10202-DPB10401, DPA10202-DPB10402, DPA10202- DPB10501, DPA10202-DPB10901, DPA10202-DPB110001, DPA10202-DPB110501, DPA10202-DPB110601, DPA10202-DPB11101, DPA10202-DPB11301, DPA10202- DPB113101, DPA10202-DPB11401, DPA10202-DPB11701, DPA10202-DPB11801, DPA10202-DPB11901, DPA10202-DPB12101, DPA10202-DPB12901, DPA10202- DPB13001, DPA10202-DPB13101, DPA10202-DPB13801, DPA10202-DPB14501, DPA10202-DPB15501, DPA10202-DPB16501, DPA10202-DPB18501, DPA10301- DPB10101, DPA10301-DPB10201, DPA10301-DPB10202, DPA10301-DPB10301, DPA10301-DPB10401, DPA10301-DPB10402, DPA10301-DPB10901, DPA10301- DPB110501, DPA10301-DPB110601, DPA10301-DPB11101, DPA10301-DPB11301, DPA10301-DPB11701, DPA10301-DPB11801, DPA10301-DPB12301, DPA10301- DPB13901, DPA10301-DPB14001, DPA10301-DPB14901, DPA10301-DPB15501, DPA10301-DPB16501, DPA10301-DPB17501, DPA10301-DPB18001, DPA10401- DPB10101, DPA10401-DPB10201, DPA10401-DPB10202, DPA10401-DPB10301, DPA10401-DPB10402, DPA10401-DPB10501, DPA10401-DPB110501, DPA10401- DPB11301, DPA10401-DPB113301, DPA10401-DPB11401, DPA10401-DPB14901, - 55 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DQA10101-DQB10501, DQA10101-DQB10601, DQA10102-DQB10501, DQA10102- DQB10502, DQA10102-DQB10503, DQA10102-DQB10601, DQA10102-DQB10602, DQA10102-DQB10604, DQA10102-DQB10609, DQA10102-DQB10611, DQA10103- DQB10501, DQA10103-DQB10503, DQA10103-DQB10601, DQA10103-DQB10602, DQA10103-DQB10609, DQA10104-DQB10501, DQA10105-DQB10501, DQA10201- DQB10301, DQA10201-DQB10302, DQA10201-DQB10303, DQA10301-DQB10301, DQA10301-DQB10302, DQA10301-DQB10303, DQA10301-DQB10304, DQA10302- DQB10301, DQA10302-DQB10302, DQA10302-DQB10303, DQA10302-DQB10304, DQA10303-DQB10301, DQA10303-DQB10302, DQA10303-DQB10303, DQA10303- DQB10304, DQA10401-DQB10301, DQA10401-DQB10319, DQA10501-DQB10201, DQA10501-DQB10203, DQA10501-DQB10301, DQA10501-DQB10319, DQA10501- DQB10501, DQA10503-DQB10301, DQA10505-DQB10201, DQA10505-DQB10202, DQA10505-DQB10301, DQA10505-DQB10302, DQA10505-DQB10309, DQA10505- DQB10319, DQA10505-DQB10402, DQA10505-DQB10501, DQA10506-DQB10303, DQA10508-DQB10301, DQA10509-DQB10301, DQA10601-DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602.

[0458] In one aspect, a method is described for formulating a composition (e.g., a vaccine), the method comprising determining that a subject in need thereof of the composition expresses one or more HLA alleles, and formulating the composition by selecting nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 835 to 1279 and SEQ ID NOs: 36797 to 61281.

[0459] In some embodiments, the one or more HLA alleles is selected from the group consisting of DPA10103-DPB10101, DPA10103-DPB10201, DPA10103-DPB10202, DPA10103-DPB10301, DPA10103-DPB10401, DPA10103-DPB10402, DPA10103- DPB10501, DPA10103-DPB10601, DPA10103-DPB11001, DPA10103-DPB110501, DPA10103-DPB11101, DPA10103-DPB112401, DPA10103-DPB112601, DPA10103- - 56 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPB11301, DPA10103-DPB113101, DPA10103-DPB113301, DPA10103-DPB11401, DPA10103-DPB11501, DPA10103-DPB11601, DPA10103-DPB11701, DPA10103- DPB11801, DPA10103-DPB11901, DPA10103-DPB12001, DPA10103-DPB12101, DPA10103-DPB12301, DPA10103-DPB12901, DPA10103-DPB13001, DPA10103- DPB13101, DPA10103-DPB13401, DPA10103-DPB13901, DPA10103-DPB14001, DPA10103-DPB14101, DPA10103-DPB14801, DPA10103-DPB14901, DPA10103- DPB15001, DPA10103-DPB15101, DPA10103-DPB15501, DPA10103-DPB15901, DPA10103-DPB18001, DPA10104-DPB10101, DPA10104-DPB10301, DPA10104- DPB11001, DPA10104-DPB11301, DPA10104-DPB11501, DPA10105-DPB10101, DPA10105-DPB10301, DPA10105-DPB10401, DPA10105-DPB11801, DPA10105- DPB15001, DPA10106-DPB10101, DPA10106-DPB11101, DPA10201-DPB10101, DPA10201-DPB10201, DPA10201-DPB10202, DPA10201-DPB10301, DPA10201- DPB10401, DPA10201-DPB10501, DPA10201-DPB10601, DPA10201-DPB110601, DPA10201-DPB11101, DPA10201-DPB11301, DPA10201-DPB113301, DPA10201- DPB11401, DPA10201-DPB11501, DPA10201-DPB11601, DPA10201-DPB11801, DPA10201-DPB11901, DPA10201-DPB13401, DPA10201-DPB14501, DPA10201- DPB15501, DPA10201-DPB19101, DPA10202-DPB10101, DPA10202-DPB10201, DPA10202-DPB10202, DPA10202-DPB10301, DPA10202-DPB10401, DPA10202- DPB10402, DPA10202-DPB10501, DPA10202-DPB10901, DPA10202-DPB110001, DPA10202-DPB110501, DPA10202-DPB110601, DPA10202-DPB11101, DPA10202- DPB11301, DPA10202-DPB113101, DPA10202-DPB11401, DPA10202-DPB11701, DPA10202-DPB11801, DPA10202-DPB11901, DPA10202-DPB12101, DPA10202- DPB12901, DPA10202-DPB13001, DPA10202-DPB13101, DPA10202-DPB13801, DPA10202-DPB14501, DPA10202-DPB15501, DPA10202-DPB16501, DPA10202- DPB18501, DPA10301-DPB10101, DPA10301-DPB10201, DPA10301-DPB10202, DPA10301-DPB10301, DPA10301-DPB10401, DPA10301-DPB10402, DPA10301- DPB10901, DPA10301-DPB110501, DPA10301-DPB110601, DPA10301-DPB11101, DPA10301-DPB11301, DPA10301-DPB11701, DPA10301-DPB11801, DPA10301- DPB12301, DPA10301-DPB13901, DPA10301-DPB14001, DPA10301-DPB14901, DPA10301-DPB15501, DPA10301-DPB16501, DPA10301-DPB17501, DPA10301- DPB18001, DPA10401-DPB10101, DPA10401-DPB10201, DPA10401-DPB10202, DPA10401-DPB10301, DPA10401-DPB10402, DPA10401-DPB10501, DPA10401- DPB110501, DPA10401-DPB11301, DPA10401-DPB113301, DPA10401-DPB11401, - 57 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPA10401-DPB14901, DQA10101-DQB10501, DQA10101-DQB10601, DQA10102- DQB10501, DQA10102-DQB10502, DQA10102-DQB10503, DQA10102-DQB10601, DQA10102-DQB10602, DQA10102-DQB10604, DQA10102-DQB10609, DQA10102- DQB10611, DQA10103-DQB10501, DQA10103-DQB10503, DQA10103-DQB10601, DQA10103-DQB10602, DQA10103-DQB10609, DQA10104-DQB10501, DQA10105- DQB10501, DQA10201-DQB10301, DQA10201-DQB10302, DQA10201-DQB10303, DQA10301-DQB10301, DQA10301-DQB10302, DQA10301-DQB10303, DQA10301- DQB10304, DQA10302-DQB10301, DQA10302-DQB10302, DQA10302-DQB10303, DQA10302-DQB10304, DQA10303-DQB10301, DQA10303-DQB10302, DQA10303- DQB10303, DQA10303-DQB10304, DQA10401-DQB10301, DQA10401-DQB10319, DQA10501-DQB10201, DQA10501-DQB10203, DQA10501-DQB10301, DQA10501- DQB10319, DQA10501-DQB10501, DQA10503-DQB10301, DQA10505-DQB10201, DQA10505-DQB10202, DQA10505-DQB10301, DQA10505-DQB10302, DQA10505- DQB10309, DQA10505-DQB10319, DQA10505-DQB10402, DQA10505-DQB10501, DQA10506-DQB10303, DQA10508-DQB10301, DQA10509-DQB10301, DQA10601- DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602.

[0460] In another aspect, an MHC class I and / or MHC class II peptide vaccine comprises nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 61281, wherein the one or more amino acid sequences encodes for one of more peptides, wherein the one or more peptides is configured to bind in vivo to one or more HLA alleles present in a subject, and wherein the one or more HLA alleles present in the subject is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, - 58 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, C1803, DPA10103-DPB10101, DPA10103-DPB10201, DPA10103- DPB10202, DPA10103-DPB10301, DPA10103-DPB10401, DPA10103-DPB10402, DPA10103-DPB10501, DPA10103-DPB10601, DPA10103-DPB11001, DPA10103- DPB110501, DPA10103-DPB11101, DPA10103-DPB112401, DPA10103-DPB112601, DPA10103-DPB11301, DPA10103-DPB113101, DPA10103-DPB113301, DPA10103- DPB11401, DPA10103-DPB11501, DPA10103-DPB11601, DPA10103-DPB11701, DPA10103-DPB11801, DPA10103-DPB11901, DPA10103-DPB12001, DPA10103- DPB12101, DPA10103-DPB12301, DPA10103-DPB12901, DPA10103-DPB13001, DPA10103-DPB13101, DPA10103-DPB13401, DPA10103-DPB13901, DPA10103- DPB14001, DPA10103-DPB14101, DPA10103-DPB14801, DPA10103-DPB14901, DPA10103-DPB15001, DPA10103-DPB15101, DPA10103-DPB15501, DPA10103- DPB15901, DPA10103-DPB18001, DPA10104-DPB10101, DPA10104-DPB10301, DPA10104-DPB11001, DPA10104-DPB11301, DPA10104-DPB11501, DPA10105- DPB10101, DPA10105-DPB10301, DPA10105-DPB10401, DPA10105-DPB11801, DPA10105-DPB15001, DPA10106-DPB10101, DPA10106-DPB11101, DPA10201- DPB10101, DPA10201-DPB10201, DPA10201-DPB10202, DPA10201-DPB10301, DPA10201-DPB10401, DPA10201-DPB10501, DPA10201-DPB10601, DPA10201- DPB110601, DPA10201-DPB11101, DPA10201-DPB11301, DPA10201-DPB113301, DPA10201-DPB11401, DPA10201-DPB11501, DPA10201-DPB11601, DPA10201- - 59 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPB11801, DPA10201-DPB11901, DPA10201-DPB13401, DPA10201-DPB14501, DPA10201-DPB15501, DPA10201-DPB19101, DPA10202-DPB10101, DPA10202- DPB10201, DPA10202-DPB10202, DPA10202-DPB10301, DPA10202-DPB10401, DPA10202-DPB10402, DPA10202-DPB10501, DPA10202-DPB10901, DPA10202- DPB110001, DPA10202-DPB110501, DPA10202-DPB110601, DPA10202-DPB11101, DPA10202-DPB11301, DPA10202-DPB113101, DPA10202-DPB11401, DPA10202- DPB11701, DPA10202-DPB11801, DPA10202-DPB11901, DPA10202-DPB12101, DPA10202-DPB12901, DPA10202-DPB13001, DPA10202-DPB13101, DPA10202- DPB13801, DPA10202-DPB14501, DPA10202-DPB15501, DPA10202-DPB16501, DPA10202-DPB18501, DPA10301-DPB10101, DPA10301-DPB10201, DPA10301- DPB10202, DPA10301-DPB10301, DPA10301-DPB10401, DPA10301-DPB10402, DPA10301-DPB10901, DPA10301-DPB110501, DPA10301-DPB110601, DPA10301- DPB11101, DPA10301-DPB11301, DPA10301-DPB11701, DPA10301-DPB11801, DPA10301-DPB12301, DPA10301-DPB13901, DPA10301-DPB14001, DPA10301- DPB14901, DPA10301-DPB15501, DPA10301-DPB16501, DPA10301-DPB17501, DPA10301-DPB18001, DPA10401-DPB10101, DPA10401-DPB10201, DPA10401- DPB10202, DPA10401-DPB10301, DPA10401-DPB10402, DPA10401-DPB10501, DPA10401-DPB110501, DPA10401-DPB11301, DPA10401-DPB113301, DPA10401- DPB11401, DPA10401-DPB14901, DQA10101-DQB10501, DQA10101-DQB10601, DQA10102-DQB10501, DQA10102-DQB10502, DQA10102-DQB10503, DQA10102- DQB10601, DQA10102-DQB10602, DQA10102-DQB10604, DQA10102-DQB10609, DQA10102-DQB10611, DQA10103-DQB10501, DQA10103-DQB10503, DQA10103- DQB10601, DQA10103-DQB10602, DQA10103-DQB10609, DQA10104-DQB10501, DQA10105-DQB10501, DQA10201-DQB10301, DQA10201-DQB10302, DQA10201- DQB10303, DQA10301-DQB10301, DQA10301-DQB10302, DQA10301-DQB10303, DQA10301-DQB10304, DQA10302-DQB10301, DQA10302-DQB10302, DQA10302- DQB10303, DQA10302-DQB10304, DQA10303-DQB10301, DQA10303-DQB10302, DQA10303-DQB10303, DQA10303-DQB10304, DQA10401-DQB10301, DQA10401- DQB10319, DQA10501-DQB10201, DQA10501-DQB10203, DQA10501-DQB10301, DQA10501-DQB10319, DQA10501-DQB10501, DQA10503-DQB10301, DQA10505- DQB10201, DQA10505-DQB10202, DQA10505-DQB10301, DQA10505-DQB10302, DQA10505-DQB10309, DQA10505-DQB10319, DQA10505-DQB10402, DQA10505- DQB10501, DQA10506-DQB10303, DQA10508-DQB10301, DQA10509-DQB10301, - 60 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DQA10601-DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602.

[0461] In one aspect, a method is described comprising administering to a subject a composition (e.g., a vaccine) in an effective amount to induce an immune response in the subject, the composition comprising nucleic acid sequences encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 61281.

[0462] In some embodiments, the method comprises administering the composition to the subject based on a determination that the subject expresses one or more HLA alleles. In some embodiments, the one or more HLA alleles is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, - 61 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, C1803, DPA10103-DPB10101, DPA10103-DPB10201, DPA10103-DPB10202, DPA10103-DPB10301, DPA10103- DPB10401, DPA10103-DPB10402, DPA10103-DPB10501, DPA10103-DPB10601, DPA10103-DPB11001, DPA10103-DPB110501, DPA10103-DPB11101, DPA10103- DPB112401, DPA10103-DPB112601, DPA10103-DPB11301, DPA10103-DPB113101, DPA10103-DPB113301, DPA10103-DPB11401, DPA10103-DPB11501, DPA10103- DPB11601, DPA10103-DPB11701, DPA10103-DPB11801, DPA10103-DPB11901, DPA10103-DPB12001, DPA10103-DPB12101, DPA10103-DPB12301, DPA10103- DPB12901, DPA10103-DPB13001, DPA10103-DPB13101, DPA10103-DPB13401, DPA10103-DPB13901, DPA10103-DPB14001, DPA10103-DPB14101, DPA10103- DPB14801, DPA10103-DPB14901, DPA10103-DPB15001, DPA10103-DPB15101, DPA10103-DPB15501, DPA10103-DPB15901, DPA10103-DPB18001, DPA10104- DPB10101, DPA10104-DPB10301, DPA10104-DPB11001, DPA10104-DPB11301, DPA10104-DPB11501, DPA10105-DPB10101, DPA10105-DPB10301, DPA10105- DPB10401, DPA10105-DPB11801, DPA10105-DPB15001, DPA10106-DPB10101, DPA10106-DPB11101, DPA10201-DPB10101, DPA10201-DPB10201, DPA10201- DPB10202, DPA10201-DPB10301, DPA10201-DPB10401, DPA10201-DPB10501, DPA10201-DPB10601, DPA10201-DPB110601, DPA10201-DPB11101, DPA10201- DPB11301, DPA10201-DPB113301, DPA10201-DPB11401, DPA10201-DPB11501, DPA10201-DPB11601, DPA10201-DPB11801, DPA10201-DPB11901, DPA10201- DPB13401, DPA10201-DPB14501, DPA10201-DPB15501, DPA10201-DPB19101, DPA10202-DPB10101, DPA10202-DPB10201, DPA10202-DPB10202, DPA10202- DPB10301, DPA10202-DPB10401, DPA10202-DPB10402, DPA10202-DPB10501, DPA10202-DPB10901, DPA10202-DPB110001, DPA10202-DPB110501, DPA10202- DPB110601, DPA10202-DPB11101, DPA10202-DPB11301, DPA10202-DPB113101, DPA10202-DPB11401, DPA10202-DPB11701, DPA10202-DPB11801, DPA10202- DPB11901, DPA10202-DPB12101, DPA10202-DPB12901, DPA10202-DPB13001, DPA10202-DPB13101, DPA10202-DPB13801, DPA10202-DPB14501, DPA10202- DPB15501, DPA10202-DPB16501, DPA10202-DPB18501, DPA10301-DPB10101, DPA10301-DPB10201, DPA10301-DPB10202, DPA10301-DPB10301, DPA10301- DPB10401, DPA10301-DPB10402, DPA10301-DPB10901, DPA10301-DPB110501, - 62 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPA10301-DPB110601, DPA10301-DPB11101, DPA10301-DPB11301, DPA10301- DPB11701, DPA10301-DPB11801, DPA10301-DPB12301, DPA10301-DPB13901, DPA10301-DPB14001, DPA10301-DPB14901, DPA10301-DPB15501, DPA10301- DPB16501, DPA10301-DPB17501, DPA10301-DPB18001, DPA10401-DPB10101, DPA10401-DPB10201, DPA10401-DPB10202, DPA10401-DPB10301, DPA10401- DPB10402, DPA10401-DPB10501, DPA10401-DPB110501, DPA10401-DPB11301, DPA10401-DPB113301, DPA10401-DPB11401, DPA10401-DPB14901, DQA10101- DQB10501, DQA10101-DQB10601, DQA10102-DQB10501, DQA10102-DQB10502, DQA10102-DQB10503, DQA10102-DQB10601, DQA10102-DQB10602, DQA10102- DQB10604, DQA10102-DQB10609, DQA10102-DQB10611, DQA10103-DQB10501, DQA10103-DQB10503, DQA10103-DQB10601, DQA10103-DQB10602, DQA10103- DQB10609, DQA10104-DQB10501, DQA10105-DQB10501, DQA10201-DQB10301, DQA10201-DQB10302, DQA10201-DQB10303, DQA10301-DQB10301, DQA10301- DQB10302, DQA10301-DQB10303, DQA10301-DQB10304, DQA10302-DQB10301, DQA10302-DQB10302, DQA10302-DQB10303, DQA10302-DQB10304, DQA10303- DQB10301, DQA10303-DQB10302, DQA10303-DQB10303, DQA10303-DQB10304, DQA10401-DQB10301, DQA10401-DQB10319, DQA10501-DQB10201, DQA10501- DQB10203, DQA10501-DQB10301, DQA10501-DQB10319, DQA10501-DQB10501, DQA10503-DQB10301, DQA10505-DQB10201, DQA10505-DQB10202, DQA10505- DQB10301, DQA10505-DQB10302, DQA10505-DQB10309, DQA10505-DQB10319, DQA10505-DQB10402, DQA10505-DQB10501, DQA10506-DQB10303, DQA10508- DQB10301, DQA10509-DQB10301, DQA10601-DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602.

[0463] In one aspect, a method is described for formulating a composition (e.g., a vaccine), the method comprising determining that a subject in need thereof of the composition expresses one or more HLA alleles, and formulating the composition by selecting nucleic acid sequences - 63 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 encoding for one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 61281.

[0464] In some embodiments, the one or more HLA alleles is selected from the group consisting of A0101, A0102, A0103, A0109, A0123, A0201, A0202, A0203, A0204, A0205, A0206, A0207, A0211, A0214, A02264, A0230, A0301, A0302, A0305, A1101, A1102, A2301, A2402, A2403, A2407, A2410, A2464, A2501, A2601, A2603, A2608, A2612, A2630, A2901, A2902, A2911, A3001, A3002, A3004, A3009, A3010, A3101, A3104, A3201, A3301, A3303, A3305, A3401, A3402, A3601, A6601, A6602, A6603, A6801, A6802, A6827, A6901, A74, A7401, A7402, A7403, A7404, A7405, A7406, A7407, A7408, A7409, A7410, A7411, A7413, A8001, B0702, B0705, B0801, B1301, B1302, B1303, B1401, B1402, B1403, B1501, B1502, B1503, B1508, B1510, B1511, B1512, B1516, B1517, B1518, B1521, B15220, B1524, B1525, B1527, B1531, B1532, B1537, B1801, B1802, B1803, B2702, B2703, B2704, B2705, B2706, B2707, B3501, B3502, B3503, B3505, B3508, B3512, B3532, B3541, B3543, B3701, B3801, B3802, B3901, B3905, B3906, B3909, B3910, B3924, B4001, B4002, B4006, B4008, B40114, B4012, B4016, B4101, B4102, B4201, B4202, B4402, B4403, B4404, B4405, B4407, B4410, B4415, B4427, B4501, B4507, B4601, B4701, B4703, B4801, B4803, B4901, B5001, B5101, B5102, B5105, B5107, B5108, B5109, B5201, B5301, B5401, B5501, B5502, B5601, B5604, B5610, B5701, B5702, B5703, B5704, B5801, B5802, B6701, B7301, B7801, B8101, B8103, B8201, B8202, C0102, C0103, C0144, C0202, C0210, C0214, C0217, C0229, C0302, C0303, C0304, C0305, C0317, C0401, C0403, C0404, C0407, C0501, C0509, C0602, C0701, C0702, C0704, C0705, C0706, C0718, C0801, C0802, C0803, C0804, C0812, C1202, C1203, C1402, C1403, C1502, C1504, C1505, C1509, C1601, C1602, C1604, C16112, C1646, C17, C1701, C1702, C1703, C1704, C1705, C1706, C1707, C18, C1801, C1802, C1803, DPA10103- DPB10101, DPA10103-DPB10201, DPA10103-DPB10202, DPA10103-DPB10301, DPA10103-DPB10401, DPA10103-DPB10402, DPA10103-DPB10501, DPA10103- DPB10601, DPA10103-DPB11001, DPA10103-DPB110501, DPA10103-DPB11101, DPA10103-DPB112401, DPA10103-DPB112601, DPA10103-DPB11301, DPA10103- DPB113101, DPA10103-DPB113301, DPA10103-DPB11401, DPA10103-DPB11501, DPA10103-DPB11601, DPA10103-DPB11701, DPA10103-DPB11801, DPA10103- DPB11901, DPA10103-DPB12001, DPA10103-DPB12101, DPA10103-DPB12301, DPA10103-DPB12901, DPA10103-DPB13001, DPA10103-DPB13101, DPA10103- DPB13401, DPA10103-DPB13901, DPA10103-DPB14001, DPA10103-DPB14101, DPA10103-DPB14801, DPA10103-DPB14901, DPA10103-DPB15001, DPA10103- - 64 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DPB15101, DPA10103-DPB15501, DPA10103-DPB15901, DPA10103-DPB18001, DPA10104-DPB10101, DPA10104-DPB10301, DPA10104-DPB11001, DPA10104- DPB11301, DPA10104-DPB11501, DPA10105-DPB10101, DPA10105-DPB10301, DPA10105-DPB10401, DPA10105-DPB11801, DPA10105-DPB15001, DPA10106- DPB10101, DPA10106-DPB11101, DPA10201-DPB10101, DPA10201-DPB10201, DPA10201-DPB10202, DPA10201-DPB10301, DPA10201-DPB10401, DPA10201- DPB10501, DPA10201-DPB10601, DPA10201-DPB110601, DPA10201-DPB11101, DPA10201-DPB11301, DPA10201-DPB113301, DPA10201-DPB11401, DPA10201- DPB11501, DPA10201-DPB11601, DPA10201-DPB11801, DPA10201-DPB11901, DPA10201-DPB13401, DPA10201-DPB14501, DPA10201-DPB15501, DPA10201- DPB19101, DPA10202-DPB10101, DPA10202-DPB10201, DPA10202-DPB10202, DPA10202-DPB10301, DPA10202-DPB10401, DPA10202-DPB10402, DPA10202- DPB10501, DPA10202-DPB10901, DPA10202-DPB110001, DPA10202-DPB110501, DPA10202-DPB110601, DPA10202-DPB11101, DPA10202-DPB11301, DPA10202- DPB113101, DPA10202-DPB11401, DPA10202-DPB11701, DPA10202-DPB11801, DPA10202-DPB11901, DPA10202-DPB12101, DPA10202-DPB12901, DPA10202- DPB13001, DPA10202-DPB13101, DPA10202-DPB13801, DPA10202-DPB14501, DPA10202-DPB15501, DPA10202-DPB16501, DPA10202-DPB18501, DPA10301- DPB10101, DPA10301-DPB10201, DPA10301-DPB10202, DPA10301-DPB10301, DPA10301-DPB10401, DPA10301-DPB10402, DPA10301-DPB10901, DPA10301- DPB110501, DPA10301-DPB110601, DPA10301-DPB11101, DPA10301-DPB11301, DPA10301-DPB11701, DPA10301-DPB11801, DPA10301-DPB12301, DPA10301- DPB13901, DPA10301-DPB14001, DPA10301-DPB14901, DPA10301-DPB15501, DPA10301-DPB16501, DPA10301-DPB17501, DPA10301-DPB18001, DPA10401- DPB10101, DPA10401-DPB10201, DPA10401-DPB10202, DPA10401-DPB10301, DPA10401-DPB10402, DPA10401-DPB10501, DPA10401-DPB110501, DPA10401- DPB11301, DPA10401-DPB113301, DPA10401-DPB11401, DPA10401-DPB14901, DQA10101-DQB10501, DQA10101-DQB10601, DQA10102-DQB10501, DQA10102- DQB10502, DQA10102-DQB10503, DQA10102-DQB10601, DQA10102-DQB10602, DQA10102-DQB10604, DQA10102-DQB10609, DQA10102-DQB10611, DQA10103- DQB10501, DQA10103-DQB10503, DQA10103-DQB10601, DQA10103-DQB10602, DQA10103-DQB10609, DQA10104-DQB10501, DQA10105-DQB10501, DQA10201- DQB10301, DQA10201-DQB10302, DQA10201-DQB10303, DQA10301-DQB10301, - 65 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 DQA10301-DQB10302, DQA10301-DQB10303, DQA10301-DQB10304, DQA10302- DQB10301, DQA10302-DQB10302, DQA10302-DQB10303, DQA10302-DQB10304, DQA10303-DQB10301, DQA10303-DQB10302, DQA10303-DQB10303, DQA10303- DQB10304, DQA10401-DQB10301, DQA10401-DQB10319, DQA10501-DQB10201, DQA10501-DQB10203, DQA10501-DQB10301, DQA10501-DQB10319, DQA10501- DQB10501, DQA10503-DQB10301, DQA10505-DQB10201, DQA10505-DQB10202, DQA10505-DQB10301, DQA10505-DQB10302, DQA10505-DQB10309, DQA10505- DQB10319, DQA10505-DQB10402, DQA10505-DQB10501, DQA10506-DQB10303, DQA10508-DQB10301, DQA10509-DQB10301, DQA10601-DQB10301, DRB1_0101, DRB1_0102, DRB1_0103, DRB1_0301, DRB1_0401, DRB1_0402, DRB1_0404, DRB1_0405, DRB1_0407, DRB1_0408, DRB1_0410, DRB1_0701, DRB1_0801, DRB1_0802, DRB1_0803, DRB1_0804, DRB1_0806, DRB1_0809, DRB1_0813, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1102, DRB1_1103, DRB1_1104, DRB1_1114, DRB1_1201, DRB1_1202, DRB1_1301, DRB1_1302, DRB1_1303, DRB1_1304, DRB1_1305, DRB1_1312, DRB1_1401, DRB1_1402, DRB1_1403, DRB1_1404, DRB1_1405, DRB1_1406, DRB1_1407, DRB1_1454, DRB1_1501, DRB1_1502, DRB1_1503, DRB1_1601, and DRB1_1602. BRIEF DESCRIPTION OF THE DRAWINGS

[0465] The following figures depict illustrative embodiments of the invention.

[0466] FIG.1 is a flow chart of a vaccine optimization method.

[0467] FIG.2 is a flow chart of a vaccine optimization method with seed set compression.

[0468] FIG.3 shows the predicted population coverage for MHC class I vaccines for single mutations in cancer-related genes. Each row describes the coverage of a vaccine for a single mutation in one of the BRAF, CTNNB1, KRAS, PIK3CA, and TP53 genes. The columns describe the predicted population coverage of each vaccine for a specific gene mutation (e.g., BRAF G466V) at varying number of peptides in the vaccine as annotated at the top of the column.

[0469] FIG.4 shows the predicted population coverage for MHC class II vaccines for single mutations in cancer-related genes. Each row describes the coverage of a vaccine for a single mutation in one of the BRAF, CTNNB1, KRAS, PIK3CA, and TP53 genes. The columns describe the predicted population coverage of each vaccine for a specific gene mutation (e.g., BRAF G466V) at varying number of peptides in the vaccine as annotated at the top of the column. - 66 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0470] FIG.5 shows predicated peptide-HLA hits by vaccine size for a KRAS G12V vaccine for the HLA diplotype HLA-A02:03, HLA-A11:01, HLA-B55:02, HLA-B58:01, HLA- C03:02, HLA-C03:03.

[0471] FIG.6 is a table showing the respective probabilities of target presentations for various mutated protein targets across different cancers.

[0472] FIG.7 is a flow chart showing a multiple target (combined) vaccine optimization method.

[0473] FIG.8 shows an example Python implementation of the MERGEMULTI function for combined vaccine design procedures. DETAILED DESCRIPTION

[0474] The practice of the embodiments disclosed herein can employ, unless otherwise indicated, conventional techniques of genetics, molecular biology, protein chemistry, computational biology, and formulation science.

[0475] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art. Definitions

[0476] The following are definitions of terms used in the present specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0477] The term “a” or “an” means at least one, unless clearly indicated otherwise. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0478] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). - 67 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0479] The term “peptide” means a chain of amino acids of any length, for example, an amino acid polymer. The peptide can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specifically indicated, peptides are indicated with the N-terminus on the left and the sequence is written from the N-terminus to the C-terminus.

[0480] The term “composition” is meant to encompass, and is not limited to, pharmaceutical compositions and nutraceutical compositions, such as a vaccine, containing drug substance (e.g., nucleic acids or peptides). The composition may also contain one or more excipients that are inactive ingredients or compounds devoid of pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of a human.

[0481] The terms “immunogenic composition,” and “immunogenic peptide composition,” mean a composition that can induce an immune response (e.g., an adaptive immune response) in a subject, unless clearly indicated otherwise. In some embodiments, the “immunogenic composition” or the “immunogenic peptide composition” is a vaccine composition. In some embodiments, the “immunogenic composition” or the “immunogenic peptide composition” can generate acquired immunity against a pathogen or disease in a subject. In some embodiments, the acquired immunity does not prevent the disease in the subject but reduces its severity. In some embodiments, the “immunogenic composition” or the “immunogenic peptide composition” (e.g., a vaccine) promotes immune system tolerance for one or more proteins.

[0482] The term “therapeutically effective amount” or “effective amount” refers to any amount that is necessary or sufficient for achieving or promoting a desired outcome. In some instances, “an effective amount” is a therapeutically effective amount. A “therapeutically effective amount” is any amount that is necessary or sufficient for promoting or achieving a desired biological response in a subject. The “effective amount” for any particular application can vary depending on such factors as the disease or condition being treated or intended to be prevented, the particular agent being administered, the size / weight of the subject, or the severity - 68 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 of the disease or condition to be treated or prevented. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without necessitating undue experimentation.

[0483] The terms “subject” and “patient” are used interchangeably herein. The terms “subject” and “subjects” refer to an animal, preferably a mammal including a nonprimate and a primate (e.g., a monkey such as a cynomolgus monkey, a chimpanzee, and a human), and a human. The term “animal” also includes, but is not limited to, companion animals such as cats and dogs; zoo animals; wild animals; farm or sport animals such as ruminants, non-ruminants, livestock and fowl (e.g., horses, cattle, sheep, pigs, turkeys, ducks, and chickens); and laboratory animals, such as rodents (e.g., mice, rats), rabbits; and guinea pigs, as well as animals that are cloned or modified, either genetically or otherwise (e.g., transgenic animals). In some embodiments, the term “subject” or “patient” refers to a human.

[0484] The terms “treating,” “treatment,” and “therapy” as used herein refer to the attempted reduction or amelioration of the progression, severity and / or duration of a disorder, or the attempted amelioration of one or more symptoms thereof resulting from the administration of one or more modalities (e.g., one or more vaccines or therapeutic agents such as a composition described herein). In some embodiments, a subject is successfully “treated” for a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.

[0485] As used herein, the terms “prevent,” “preventing,” “prevention,” “alleviate,” or “alleviating” refer to the prevention, inhibition, or lessening of the recurrence, onset, or development of a disorder or a symptom thereof in a subject resulting from the administration of a therapy (e.g., a vaccine), or the administration of a combination of therapies (e.g., a combination of vaccine(s) and / or therapeutic agents).

[0486] Units, prefixes, and symbols are denoted in their Système International d’Unités (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value (i.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth. - 69 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Composition / Vaccine Design Considerations

[0487] In some embodiments, the disclosure provides for compositions (e.g., vaccines) that incorporate peptide sequences that will be displayed by Major Histocompatibility Complex (MHC) molecules on cells and train the immune system to recognize cancer or pathogen diseased cells. The terms MHC and HLA are used interchangeably herein to denote Major Histocompatibility Complex molecules without restriction to species. In some embodiments, the disclosure provides for compositions (e.g., vaccines) that incorporate peptide sequences that will be displayed by MHC molecules on cells to induce therapeutic tolerance in antigen-specific immunotherapy for autoimmune diseases (Alhadj Ali et al., 2017, Gibson, et al.2015). In some embodiments, a composition (e.g., a vaccine) comprises one or more peptides. In some embodiments, a composition (e.g., a vaccine) includes an RNA (e.g., mRNA) or DNA construct administered for expression in vivo that encodes for one or more peptides.

[0488] The immunogenic compositions and methods described herein are applicable for designing and preparing a broad range of compositions including vaccine compositions.

[0489] The term peptide-HLA binding is defined to be the binding of a peptide to an HLA allele, and can either be computationally predicted, experimentally observed, or computationally predicted using experimental observations. The metric or score of peptide-HLA binding can be expressed as affinity (for example, based on the equilibrium dissociation constant (KD), measured in molar units (M)), percentile rank, binary at a predetermined threshold, probability, rate of disassociation, or other metrics as are known in the art. The term peptide display or peptide-HLA display describes the binding of a peptide to an HLA allele on the surface of a cell. Peptide binding to an HLA allele is required for peptide display by that HLA allele. The metric or score of peptide-HLA display can be expressed as affinity (for example, based on the equilibrium dissociation constant (KD), measured in molar units (M)), percentile rank, binary at a predetermined threshold, probability, or other metrics as are known in the art. In some embodiments, metrics of peptide-HLA binding are used for metrics of peptide-HLA display since peptide-HLA display depends upon peptide-HLA binding.

[0490] The term peptide-HLA immunogenicity metric is defined as the activation of T cells based upon their recognition of a peptide when bound by an HLA allele. The term peptide-HLA immunogenicity score is another term for a peptide-HLA immunogenicity metric, and the terms are interchangeable. A peptide-HLA immunogenicity metric can vary from individual to individual, and the metric for peptide-HLA immunogenicity can be expressed as a probability, a - 70 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 binary indicator, or other metric that relates to the likelihood that a peptide-HLA combination will be immunogenic. In some embodiments, peptide-HLA immunogenicity is defined as the induction of immune tolerance based upon the recognition of a peptide when bound by an HLA allele. A peptide-HLA immunogenicity metric can be computationally predicted, experimentally observed, or computationally predicted using experimental observations. In some embodiments, a peptide-HLA immunogenicity metric is based only upon peptide-HLA binding, since peptide-HLA binding is necessary for peptide-HLA immunogenicity. In some embodiments, peptide-HLA immunogenicity data or computational predictions of peptide-HLA immunogenicity can be included and combined with scores for peptide binding or peptide display in the methods disclosed herein. One way of combining the scores is using immunogenicity data for peptides assayed for immunogenicity in diseased or vaccinated individuals and assigning peptides to the HLA allele that displayed them in the individual by choosing the HLA allele that computational methods predict has the highest likelihood of display. For peptides that are not experimentally assayed, computational predictions of binding can be used. In some embodiments, different computational methods of predicting peptide-HLA immunogenicity or peptide-HLA binding can be combined (Liu et al., 2020b). For a given set of peptides and a set of HLA alleles, the term peptide-HLA hits is the number of unique combinations of peptides and HLA alleles that exhibit peptide-HLA immunogenicity or binding at a predetermined threshold. For example, a peptide-HLA hit of 2 can mean that one peptide is predicted to be bound (or trigger T cell activation) by two different HLA alleles, two peptides are predicted to be bound (or trigger T cell activation) by two different HLA alleles, or two peptides are predicted to be bound (or trigger T cell activation) by the same HLA allele. For a given set of peptides and HLA frequencies, HLA haplotype frequencies, or HLA diplotype frequencies, the expected number of peptide-HLA hits is the average number of peptide-HLA hits in each set of HLAs that represent an individual, weighted by their frequency of occurrence.

[0491] Peptide display by an MHC molecule is necessary, but not sufficient, for a peptide to be immunogenic and cause the recognition of the resulting peptide-MHC complex by an individual’s T cells to trigger T cell activation, expansion, and immune memory. In some embodiments, ELISPOT (Slota et al., 2011) or the Multiplex Identification of Antigen-Specific T Cell Receptors Using a Combination of Immune Assays and Immune Receptor Sequencing (MIRA) assay (Klinger et al., 2015) are used to score peptide display or affinity (e.g., a peptide immunogenicity that requires peptide binding) by an MHC molecule (e.g., HLA allele, measured as a peptide-HLA binding score). In some embodiments, experimental data from - 71 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 assays such as the ELISPOT (Slota et al., 2011) or the MIRA assay can be used to produce a peptide-HLA immunogenicity metric with respect to a peptide and an HLA allele in a given experimental context or individual. In some embodiments, experimental data from assays such as the ELISPOT (Slota et al., 2011) or the MIRA assay can be combined with machine learning based predictions for scoring peptide display by an MHC molecule or peptide binding (e.g., binding affinity) by an MHC molecule (e.g., HLA allele, measured as a peptide-HLA binding score) for determining a peptide-HLA immunogenicity metric. In some embodiments, the MHCflurry or NetMHCpan (Reynisson et al., 2020) computational methods are used to predict MHC class I display of a peptide by an HLA allele. In some embodiments, the NetMHCIIpan computational method (Reynisson et al., 2020) is used to predict MHC class II display of a peptide by an HLA allele (see Table 1).

[0492] In some embodiments, a composition (e.g., an immunogenic composition) includes nucleic acids sequences encoding for one or more peptides, wherein the one or more peptides is MHC restricted (also referred to as HLA restricted). In some embodiments, a composition (e.g., an immunogenic composition) includes one or more peptides, wherein the one or more peptides is MHC restricted (also referred to as HLA restricted). In some embodiments, an MHC restriction of a peptide sequence refers to the presence of an MHC (or HLA) allele in a subject that allows for peptide-HLA display, peptide-HLA binding, or peptide-HLA immunogenicity for the peptide. An HLA allele is abbreviated by its locus, its two digit allele group, and its two or three digit specific HLA protein. For example, the HLA allele HLA-A*02:01 is abbreviated as A0201. For MHC Class II alleles that begin with DP, DQ, or DR, the first digit is part of the locus, followed by a two or three digit allele group and a two or three digit specific HLA protein. For example, the HLA allele DPA1*01:03 is abbreviated DPA10103 and DRB1*15:01 is abbreviated DRB1_1501. In some embodiments, a composition is designed based on MHC restrictions such that one or more peptides (or nucleic acids encoding for one or more peptides) is included in the composition based on a determination that one or more MHC (or HLA) alleles is present in a subject in need thereof of the composition. In some embodiments, the MHC (or HLA) alleles present in the subject are determined to allow for display or immunogenicity (peptide-HLA display or peptide-HLA immunogenicity) of the one or more peptides (or nucleic acids encoding for one or more peptides) in the composition. In some embodiments, the HLA alleles present or expressed in a subject are used to select one or more peptides (or nucleic acids encoding the peptides) for inclusion in a composition to be administered to the subject. In some embodiments, the selection is based on the likelihood that a given HLA allele expressed in the - 72 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 subject is capable of binding to a given peptide (or nucleic acid encoding the peptide) in the composition. In some embodiments, a composition is designed based on MHC restrictions such that one or more peptides (or nucleic acids encoding for one or more peptides) is included in the composition based on a determination of the MHC (or HLA) alleles present in a desired population of people. Population based HLA allele frequencies can be determined from the Allele Frequency Net Database, the HLA haplotype frequencies provided in Liu et al., Cell Systems 11, Issue 2, p.131-146 (Liu et al., 2020a), or other sources known in the art. In some embodiments, algorithms such as OptiVax are used for peptide selection using population HLA frequencies (Liu et al., 2020a, Liu et al., 2022). A given peptide sequence can have more than one MHC restriction. For example, SEQ ID NO: 16 has an MHC restriction that includes an HLA allele selected from the group consisting of A3201, B1517, and B1525. In some embodiments, a peptide (or nucleic acid sequences encoding for the peptide) is included in a composition only if two or more MHC (or HLA) alleles are present the subject that are also in the MHC restriction of the peptide. For example, the peptide (or nucleic acid sequences encoding for the peptide) containing SEQ ID NO: 16 is included in the composition only if a subject has both A3201 and B1517 alleles. In some embodiments, MHC restrictions can be based on both MHC class I and MHC class II alleles.

[0493] In some embodiments, the one or more amino acid sequences is selected for inclusion in a composition (e.g., a vaccine) based on an MHC restriction that includes an HLA allele described in the notes field of the respective SEQ ID NO entry in the sequence listing. For example, in the sequence listing, SEQ ID NO: 16 has a note filed that states: “HLAs: A3201 B1517 B1525.” Thus, in some embodiments, SEQ ID NO:16 is included in the composition if it is determined that an individual in need thereof of the composition expresses one or more of the HLA alleles selected from the group consisting of A3201, B1517, and B1525. Other sequences listed in the sequence listing are considered for inclusion in a composition based on their corresponding HLA alleles as listed in their respective notes field. In some embodiments, the one or more amino acid sequences is selected for inclusion in the MHC class II peptide vaccine based on an MHC restriction that includes an HLA allele described in the notes field of the respective SEQ ID NO entry in the sequence listing. For example, SEQ ID NO: 835 has an MHC restriction that includes an HLA allele selected from the group consisting of DRB1_0101, DRB1_0701, DRB1_0901, DRB1_1001, DPA10103-DPB10601, DPA10103-DPB15001, DPA10105-DPB15001, DPA10401-DPB10301, DPA10401-DPB10501, DPA10401- DPB11301, DPA10401-DPB113301, and DPA10401-DPB11401. In some embodiments, the - 73 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 one or more amino acid sequences is selected for inclusion in the MHC class I and / or MHC class II peptide vaccine based on an MHC restriction that includes an HLA allele described in the notes field of the respective SEQ ID NO entry in the sequence listing.

[0494] Non-limiting MHC restrictions for entries in the sequence listing are provided in a sequence’s entry free text. In some embodiments, a peptide sequence is preferably selected for inclusion in a composition based on an individual’s HLA type that is included in the sequences’ non-limiting annotated MHC restrictions. For example, SEQ ID NO: 16 has a non-limiting MHC restriction that includes HLA alleles selected from the group consisting of A3201, B1517, and B1525, and thus in some embodiments SEQ ID NO: 16 would be included in peptide candidates for a vaccine for an individual where one or more of these HLA alleles is present.

[0495] In some embodiments, computational methods such as MHCflurry (O’Donnell et al., 2018, O’Donnell et al., 2020, incorporated by reference in their entireties herein), NetMHCpan (Reynisson et al., 2020, incorporated by reference in its entirety herein), and NetMHCIIpan (Reynisson et al., 2020) are used to predict either MHC class I (MHCflurry, NetMHCpan) or class II (NetMHCIIpan) display of peptides by an HLA allele. In other embodiments, other methods of determining peptide-HLA binding are used as disclosed in International Publication No. WO 2005 / 042698, incorporated by reference in its entirety herein. NetMHCpan-4.1 and NetMHCIIpan-4.0 utilize the NNAlign_MA algorithm (Alvarez et al., 2019, incorporated by reference in its entirety herein) for predicting peptide-HLA binding. NNAlign_MA is in turn based upon the NNAlign (Nielsen et al., 2009, Nielsen et al., 2017, incorporated by reference in their entireties herein) neural network. NetMHCpan-4.1 (Reynisson et al., 2020) uses NNAlign_MA networks with at least 180 one-hot encoded inputs that describe the peptide sequence (9 residues x 20 possible amino acids per residue = 180 inputs). Networks with both 56 and 66 hidden neurons and two outputs are utilized (Alvarez et al., 2019). One output produces a binding affinity data type and the other output produces a mass spectrometry based eluted ligand data type (Alvarez et al., 2019). In some embodiments, the binding affinity data type is used as a peptide-HLA binding metric. In some embodiments, the binding affinity data type is used as a peptide-HLA display metric. In some embodiments, the eluted ligand data type output is used as a peptide-HLA binding metric. In some embodiments, the eluted ligand data type output is used as a peptide-HLA display metric. In some embodiments, the binding affinity data type is used as a peptide-HLA immunogenicity metric. In some embodiments, the eluted ligand data type is used as a peptide-HLA immunogenicity metric. Each network architecture - 74 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 (56 or 66 hidden neurons) is trained with 5 different random parameter initializations and 5-fold cross-validation resulting in a total of 50 individual trained networks (2 architectures x 5 initializations x 5 cross-validation). These 50 trained networks are used as an ensemble with 25 networks having at least 10,800 parameters (180 inputs x 56 neurons) and 25 networks consist of at least 11,880 parameters (180 inputs x 66 neurons). Thus, the ensemble of 50 networks in NetMHCpan-4.1 consists of at least 567,000 parameters that must be evaluated with at least 567,000 arithmetic operations for computing peptide-MHC binding. NetMHCIIpan-4.1 (Reynisson et al., 2020) uses NNAlign_MA networks with at least 180 inputs that describe the peptide sequence (9 x 20 = 180 inputs). Networks with 2, 10, 20, 40, and 60 hidden neurons and two outputs are utilized (Alvarez et al., 2019). Each network architecture (2, 10, 20, 40, or 60 hidden neurons) is trained with 10 different random parameter initializations and 5-fold cross- validation resulting in a total of 250 individual trained networks (5 architectures x 10 initializations x 5 cross-validation). These 250 trained networks are used as an ensemble with 50 networks having at least 360 parameters (180 inputs x 2 neurons), 50 networks having at least 1800 parameters (180 inputs x 10 neurons), 50 networks having at least 3600 parameters (180 inputs x 20 neurons), 50 networks having at least 7200 parameters (180 inputs x 40 neurons), and 50 networks having at least 10,800 parameters (180 inputs x 60 neurons). Thus, the ensemble of 250 networks in NetMHCIIpan-4.0 consists of at least 1,188,000 parameters that must be evaluated with at least 1,188,000 arithmetic operations for computing peptide-MHC binding.

[0496] In some embodiments, computational methods used to predict either MHC class I (e.g., MHCflurry, NetMHCpan) or class II (e.g., NetMHCIIpan) peptide-HLA binding scores or peptide-HLA immunogenicity metrics are based upon data from experimental mass spectrometry observations of peptides bound by MHC molecules. In some embodiments, computational methods used to predict either MHC class I (e.g., MHCflurry, NetMHCpan) or class II (e.g., NetMHCIIpan) peptide-HLA binding scores or peptide-HLA immunogenicity metrics are based upon data from experimental observations of peptide-MHC binding affinity. In some embodiments, experimental observations of peptide-MHC binding affinity or immunogenicity, including mass spectrometry measurements of peptide-HLA binding and measurements of T cell activation, can be found in databases such as the Immune Epitope Database (IEDB) (Vita et al., 2018). The output of MHCflurry 2.0 (O’Donnell et al., 2020, incorporated by reference in its entirety herein) is based upon 493,473 mass spectrometry measurements of peptide-HLA binding, and 219,596 affinity measurements of peptide-HLA - 75 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 binding. The output of NetMHCpan-4.1 (Reynisson et al., 2020) is based upon 665,492 mass spectrometry measurements of peptide-HLA binding, and 52,402 affinity measurements of peptide-HLA binding. The output of NetMHCIIpan-4.0 (Reynisson et al., 2020) is based upon 381,066 mass spectrometry measurements of peptide-HLA binding, and 44,861 affinity measurements of peptide-HLA binding.

[0497] A peptide is displayed by an MHC molecule when it binds within the groove of the MHC molecule and is transported to the cell surface where it can be recognized by a T cell receptor. A target peptide refers to a foreign peptide or a self-peptide. In some embodiments, a peptide that is part of the normal proteome in a healthy individual is a self-peptide, and a peptide that is not part of the normal proteome is a foreign peptide. In some embodiments, target peptides can be part of the normal proteome that exhibit aberrant expression (e.g., cancer-testis antigens such as NY-ESO-1). Foreign peptides can be generated by mutations in normal self- proteins in tumor cells that create epitopes called neoantigens, or by pathogenic infections. In some embodiments, a neoantigen is any subsequence of a human protein, where the subsequence contains one or more altered amino acids or protein modifications that do not appear in a healthy individual. Therefore, in this disclosure, foreign peptide refers to an amino acid sequence encoding a fragment of a target protein / peptide (or a full-length protein / peptide), the target protein / peptide consisting of a neoantigen protein, a pathogen proteome, or any other undesired protein that is non-self and is expected to be bound and displayed by an HLA allele.

[0498] Protein genes identified by their UniProt ID that are frequently mutated in cancer include RASK_HUMAN (also called KRAS), AKT1_HUMAN, BRAF_HUMAN, CTNB1_HUMAN (also called CTNNB1), EGFR_HUMAN, GTF2I_HUMAN, RASH_HUMAN (also called HRAS), IDHC_HUMAN (also called IDH1), RASN_HUMAN (also called NRAS), PIK3CA_HUMAN, PTEN_HUMAN, and P53_HUMAN (also called TP53). We describe a missense mutation in a protein by the one letter amino acid code for the wild-type amino acid, the amino acid position of the mutation, and the one letter amino acid code that is present in the mutated protein. For example, KRAS G12D is a mutation in the KRAS protein of position 12 from glycine to aspartic acid (G12D). Proteins may contain multiple mutations at different positions. Herein we may refer to a gene without the “_HUMAN” suffix for conciseness.

[0499] KRAS gene mutations are the most frequently mutated oncogenes in cancer, but they have been very difficult to treat with small molecule therapeutics. The KRAS protein is part of - 76 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 a signaling pathway that controls cellular growth and point mutations in the protein can cause constitutive pathway activation and uncontrolled cell growth. Single amino acid KRAS mutations result in minor changes in protein structure, making it difficult to engineer small molecule drugs that recognize a mutant specific binding pocket and inactivate KRAS signaling. KRAS oncogenic mutations include the mutation of position 12 from glycine to aspartic acid (G12D), glycine to valine (G12V), glycine to arginine (G12R), or glycine to cystine (G12C); or the mutation of position 13 from glycine to aspartic acid (G13D). The corresponding foreign peptides contain these mutations. KRAS is a member of the RAS family of genes that also includes HRAS and NRAS. KRAS, HRAS, and NRAS have identical sequences from residue 1 to residue 86. Thus, all of the vaccines and associated peptide sequences described herein for a mutation in one RAS family member can be used for the identical mutation in any other RAS family member (e.g., a KRAS G12D vaccine is also a vaccine for HRAS G12D).

[0500] Certain self-proteins, such as cancer-testis antigens, are present in cancerous cells at aberrantly high levels and thus can be targets for vaccination to induce an intolerant T cell response against cells displaying peptides derived from these self-proteins on MHC molecules. Examples of these cancer-related proteins by their UniProt IDs include CTG1B_HUMAN (also known as NY-ESO-1), MAGA1_HUMAN, MAGA3_HUMAN, MAGA4_HUMAN, MAGC1_HUMAN, MAGC3_HUMAN, SSX2_HUMAN, PRAME_HUMAN, KKLC1_HUMAN (also known as CT83), PMEL_HUMAN (as known as gp100), TYRP1_HUMAN (also known as gp75), TYRP2_HUMAN (also known as DCT), and MAR1_HUMAN.

[0501] Autoimmune disorders are caused by the loss of self-tolerance by the immune system to self-proteins and are involved in autoimmune disorders such as diabetes, multiple sclerosis, and autoimmune encephalomyelitis. Induction of tolerance for autoimmune-related self- peptides can be accomplished by antigen-specific tolerization using the delivery of vaccine antigens with a tolerization protocol. An example of a protocol for the induction of tolerance with a lipid-nanoparticle (LNP) encapsulating RNA (e.g., mRNA-LNP) vaccine is described by Krienke et al., 2021 and is incorporated by reference in its entirety herein. Examples of autoimmune disease-related proteins include UniProt IDs INS_HUMAN (also known as insulin), and MOG_HUMAN (also known as Myelin-oligodendrocyte glycoprotein). Individuals with diabetes can suffer from a lack of tolerance to INS_HUMAN, and individuals - 77 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 with multiple sclerosis or autoimmune encephalomyelitis can suffer from a lack of tolerance to MOG_HUMAN.

[0502] A challenge for the design of peptide vaccines is the diversity of human MHC alleles (HLA alleles) that each have specific preferences for the peptide sequences they will display. The Human Leukocyte Antigen (HLA) loci, located within the MHC, encode the HLA class I and class II molecules. There are three classical class I loci (HLA-A, HLA-B, and HLA-C) and three loci that encode class II molecules (HLA-DR, HLA-DQ, and HLA-DP). An individual’s HLA type describes the alleles they carry at each of these loci. Peptides of length of between about 8 and about 11 residues can bind to HLA class I (or MHC class I) molecules whereas those peptides of length of between about 13 and about 25 residues bind to HLA class II (or MHC class II) molecules (Rist et al., 2013; Chicz et al., 1992). Human populations that originate from different geographies have differing frequencies of HLA alleles, and these populations exhibit linkage disequilibrium between HLA loci that result in population specific haplotype frequencies. In some embodiments, methods are disclosed for creating effective vaccines that include consideration of the HLA allelic frequency in the target population, as well as linkage disequilibrium between HLA genes to achieve a set of peptides that is likely to be robustly displayed.

[0503] The present disclosure provides for compositions, systems, and methods of vaccine designs that produce immunity to single or multiple targets. In some embodiments, a target is a neoantigen protein sequence, a pathogen proteome, or any other undesired protein sequence that is non-self and is expected to be bound and displayed by an HLA molecule (also referred to herein as an HLA allele). When a target is present in an individual, it may result in multiple peptide sequences that are displayed by a variety of HLA alleles. In some embodiments, it may be desirable to create a vaccine that includes selected self-peptides, and thus these selected self- peptides are considered to be the target peptides for this purpose.

[0504] Because immunogenicity may vary from individual to individual, one method to increase the probability of vaccine efficacy is to use a diverse set of target peptides (e.g., at least two peptides) to increase the chances that some subset of them will be immunogenic in a given individual. Prior research using mouse models has shown that most MHC displayed peptides are immunogenic, but immunogenicity varies from individual to individual as described in Croft et al. (2019). In some embodiments, experimental peptide-HLA immunogenicity data are used - 78 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 to determine which target peptides and their modifications will be effective immunogens in a vaccine.

[0505] Considerations for the design of peptide vaccines are outlined in Liu et al., Cell Systems 11, Issue 2, p.131-146 (Liu et al., 2020a) and Liu et al., Cell Systems 12, Issue 1, p. 102-107 (Liu et al., 2020b) and U.S. Patent No.11,058,751 and U.S. Patent No.11,161,892, which are incorporated by reference in their entireties herein.

[0506] Certain target peptides may not bind with high affinity to a wide range of HLA molecules. To increase the binding of target peptides to HLA molecules, their amino acid composition can be altered to change one or more anchor residues or other residues. In some embodiments, to increase the immunogenicity of a target peptide when displayed by HLA molecules, a target peptide’s amino acid composition can be altered to change one or more residues. Anchor residues are amino acids that interact with an HLA molecule and have the largest influence on the affinity of a peptide for an HLA molecule. Peptides with one or more altered amino acid residues are called heteroclitic peptides. In some embodiments, heteroclitic peptides include target peptides with residue modifications at anchor positions. In some embodiments, heteroclitic peptides include target peptides with residue modifications at non- anchor positions. In some embodiments, heteroclitic peptides include target peptides with residue modifications that include unnatural amino acids and amino acid derivatives. Modifications to create heteroclitic peptides can improve the binding of peptides to both MHC class I and MHC class II molecules, and the modifications required can be both peptide and MHC class specific. Since peptide anchor residues face the MHC molecule groove, they are less visible than other peptide residues to T cell receptors. Thus, heteroclitic peptides with anchor residue modifications have been observed to induce a T cell response where the stimulated T cells also respond to unmodified peptides. It has been observed that the use of heteroclitic peptides in a vaccine can improve a vaccine’s effectiveness (Zirlik et al., 2006). In some embodiments, the immunogenicity of heteroclitic peptides are experimentally determined and their ability to activate T cells that also recognize the corresponding base (also called seed) peptide of the heteroclitic peptide is determined, as is known in the art (Houghton et al., 2007). In some embodiments, these assays of the immunogenicity and cross-reactivity of heteroclitic peptides are performed when the heteroclitic peptides are displayed by specific HLA alleles. - 79 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Peptide vaccines to induce immunity to one or more targets

[0507] In some embodiments, a method is provided for formulating peptide vaccines using a single vaccine design for one or more targets. In some embodiments, a single target is a foreign protein with a specific mutation (e.g., KRAS G12D). In some embodiments, a single target is a self-protein (e.g., a protein that is overexpressed in tumor cells such as cancer / testis antigens). In some embodiments, a single target is a pathogen protein (e.g., a protein contained in a viral proteome). In some embodiments, multiple targets can be used (e.g., both KRAS G12D and KRAS G13D).

[0508] In some embodiments, the method includes extracting peptides to construct a candidate set from all target proteome sequences (e.g., entire KRAS G12D protein) as described in Liu et al. (2020a).

[0509] FIGs.1 and 2 depict flow charts, for example, vaccine design methods that can be used for MHC class I or MHC class II vaccine design. A Candidate Peptide Set (see FIGs.1 and 2) is comprised of target peptides extracted by windowing an input protein sequence. In some embodiments, extracted target peptides are of amino acid length of between about 8 and about 10 (e.g., for MHC class I binding (Rist et al., 2013)). In some embodiments, the extracted target peptides presented by MHC class I molecules are longer than 10 amino acid residues, such as 11 residues (Trolle et al., 2016). In some embodiments, extracted target peptides are of length between about 13 and about 25 (e.g., for class II binding (Chicz et al., 1992)). In some embodiments, sliding windows of various size ranges described herein are used over the entire proteome. In some embodiments, other target peptide lengths for MHC class I and class II sliding windows can be utilized. In some embodiments, computational predictions of proteasomal cleavage are used to filter or select peptides in the candidate set. One computational method for predicting proteasomal cleavage is described by Nielsen et al. (2005). In some embodiments, peptide mutation rates, glycosylation, cleavage sites, or other criteria can be used to filter peptides as described in Liu et al. (2020a). In some embodiments, peptides can be filtered based upon evolutionary sequence variation above a predetermined threshold. Evolutionary sequence variation can be computed with respect to other species, other pathogens, other pathogen strains, or other related organisms. In some embodiments, a first peptide set is the candidate set.

[0510] As shown in FIGs.1-2, in some embodiments, the next step of the method includes scoring the target peptides in the candidate set for peptide-HLA binding to all considered HLA - 80 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 alleles as described in Liu et al. (2020a) and Liu et al. (2020b). In some embodiments, a first peptide set is the candidate set after scoring the target peptides. Scoring can be accomplished for human HLA molecules, mouse H-2 molecules, swine SLA molecules, or MHC molecules of any species for which prediction algorithms are available or can be developed. Thus, vaccines targeted at non-human species can be designed with the method. Scoring metrics can include the affinity for a target peptide to an HLA allele in nanomolar, eluted ligand, presentation, and other scores that can be expressed as percentile rank or any other metric. The candidate set may be further filtered to exclude peptides whose predicted binding cores do not contain a particular pathogenic or neoantigen target residue of interest or whose predicted binding cores contain the target residue in an anchor position. The candidate set may also be filtered for target peptides of specific lengths, such as length 9 for MHC class I, for example. In some embodiments, scoring of target peptides is accomplished with experimental data or a combination of experimental data and computational prediction methods. When computational models are unavailable to make peptide-HLA binding predictions for particular (peptide, HLA) pairs, the binding value for such pairs can be defined by the mean, median, minimum, or maximum immunogenicity value taken over supported pairs, a fixed value (such as an indication of no binding), or inferred using other techniques, including a function of the prediction of the most similar (peptide, HLA) pair available in the scoring model.

[0511] In some embodiments, a base set (also referred to as seed set herein) is constructed by selecting peptides from the scored candidate set using individual peptide-HLA binding or immunogenicity criteria (e.g., first peptide set) (FIG.1). In some embodiments, since a given peptide has multiple peptide-HLA scores, the selection can be based on the peptide-HLA binding score or peptide-HLA immunogenicity metric with the best affinity or highest immunogenicity (e.g., predicted to bind the strongest or activate T cells the most for a given HLA allele). The criteria used for scoring peptide-HLA binding during the scoring procedure can accommodate different goals during the base set selection and vaccine design phases. For example, a target peptide with peptide-HLA binding affinities of 500 nM may be displayed by an individual that is diseased, but at a lower frequency than a target peptide with a 50 nM peptide-HLA binding affinity. During the combinatorial design phase of a vaccine, a more constrained affinity criteria may be used (e.g., when selecting a third peptide set, the Vaccine for Target(s) in FIGs.1 and 2), such a 50 nM, to increase the probability that a vaccine peptide will be found and displayed by HLA molecules. In some embodiments, a relatively less constrained threshold (e.g., less than about 1000 nM or less than about 500 nM) of peptide-HLA - 81 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 immunogenicity or peptide-HLA binding is used as a first threshold for filtering candidate peptide-HLA scores (the first Peptide Scoring and Score Filtering step in FIGs.1 and 2) and a relatively more constrained second threshold (e.g., less than about 50 nM) is used for filtering expanded set peptide-HLA scores (the second Peptide Filtering and Scoring step in FIGs.1 and 2) for their scores for specific HLA alleles. In some embodiments, specific peptide-HLA scores are not used for modified peptides for a given HLA for vaccine design when their unmodified counterpart peptide does not pass the first less constrained threshold. Filtering of peptide-HLA scores can occur for any relevant metric (binding affinity, probability of binding, probability of immunogenicity, etc.). This filtering of peptide-HLA scores is based on the observation that peptides that are not immunogenic enough for vaccine inclusion may be antigenic (meet the first filtering threshold) and thus recognized by T cell clonotypes expanded by a vaccine. A peptide is antigenic when it is recognized by a T cell receptor and results in a response such as CD8+ T cell cytotoxicity or CD4+ cell activation. Derivatives of an antigenic peptide may be strongly immunogenic, included in a vaccine, and thus activate and expand T cells that recognize the antigenic peptide. The expansion of T cells that recognize an unmodified antigenic peptide can provide an immune response that contributes to disease control. In some embodiments, at the first Peptide Scoring and Score Filtering step in FIGs.1 and 2 the first less constrained threshold for admitting a peptide-HLA score for a peptide for an HLA allele is determined by the best peptide-HLA score of the peptide’s heteroclitic derivatives for the same HLA. In some embodiments, the probability threshold for binding or immunogenicity for a first threshold for a peptide-HLA score may be lower for an HLA allele when the probability of immunogenicity or binding for the peptide’s best derivative is higher for the same HLA. In some embodiments, the product (or other function) of a peptide’s peptide-HLA binding or immunogenicity probability score and the peptide-HLA binding or immunogenicity probability score for its best derivative peptide for the same HLA allele are required to meet a specified threshold (e.g., 0.5, 0.6, 0.7, 0.8, or 0.9). In some embodiments, peptides are scored for third peptide set (Vaccine for Target(s) in FIGs.1 and 2) potential inclusion that have peptide-HLA binding affinities less than about 500 nM. In some embodiments, peptides are selected for the base set that have peptide- HLA binding affinities less than about 1000 nM for at least one HLA allele. Alternatively, predictions of peptide-HLA immunogenicity can be used to qualify target peptides for base set inclusion. In some embodiments, experimental observations of the immunogenicity of peptides in the context of their display by HLA alleles or experimental observation of the binding of - 82 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 peptides to HLA alleles can be used to score peptides for binding to HLA alleles or peptide- HLA immunogenicity.

[0512] In some embodiments, experimental observations of the display of peptides by specific HLA alleles in tumor cells can be used to score peptides for peptide-HLA binding or peptide-HLA immunogenicity. In some embodiments, experimental observations of the display of peptides tumor cells by a specific HLA allele can be used to score peptides for peptide-HLA binding or peptide-HLA immunogenicity for that HLA allele. In some embodiments, experimental observations of the display of peptides tumor cells can be used to score peptides for peptide-HLA binding or peptide-HLA immunogenicity, with the HLA allele(s) for a specific observed peptide selected from the HLA alleles present in the tumor that meet a predicted peptide-HLA binding or immunogenicity threshold. In some embodiments, mass spectrometry is used to experimentally determine the display of peptides by tumor cells as described by Bear et al. (2021) or Wang et al. (2019) and these data are used to score for peptide-HLA binding or peptide-HLA immunogenicity. In some embodiments, mass spectrometry is used to experimentally determine the display of peptides by tumor cells, and these experimental data are used to qualify the inclusion of base set (seed set) peptides for one or more HLA alleles for a vaccine. In some embodiments, mass spectrometry is used to experimentally determine the display of a peptide by tumor cells, and these experimental data are used to exclude peptide- HLA binding scores or peptide-HLA immunogenicity scores for the peptide when the peptide is not observed to be displayed by an HLA allele by mass spectrometry. In some embodiments, mass spectrometry is used to experimentally determine the display of peptides by tumor cells in an individual, and these experimental data are used to qualify the inclusion of base set (seed set) peptides for that individual for one or more HLA alleles. In some embodiments, mass spectrometry is used to experimentally determine the display of a peptide by tumor cells in an individual, and these experimental data are used to exclude peptide-HLA binding scores or peptide-HLA immunogenicity scores for the peptide when the peptide is not observed to be displayed by an HLA allele by mass spectrometry. In some embodiments, computational predictions of the immunogenicity of a peptide in the context of display by HLA alleles can used for scoring such as the methods of Ogishi et al. (2019) or Bulik-Sullivan et al. (2019).

[0513] In some embodiments, a peptide-HLA score or a peptide-HLA immunogenicity score for a first peptide in the base set (seed set) for a given HLA allele is eliminated and not considered during vaccine design if the wild-type peptide corresponding to the first peptide (e.g., - 83 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 the unmutated naturally occurring form for the peptide or a peptide in the respective species within a defined sequence edit distance) has a peptide-HLA score or a peptide-HLA immunogenicity score for the same HLA allele within a defined threshold. The threshold can be based upon the difference of the scores of the first peptide and the wild-type peptide, the ratio of the scores of the first peptide and the wild-type peptide, the score of the wild-type peptide, or other metrics. The defined threshold can be either greater than or less than a specified value. In some embodiments, the threshold is defined so that the wild-type peptide is not predicted to be presented. In some embodiments, when a peptide-HLA score or peptide-HLA immunogenicity score is eliminated for a first peptide during vaccine design, then peptide-HLA scores or peptide-HLA immunogenicity scores for all of its derivatives (e.g., heteroclitic peptide derivatives) for the same HLA allele are also eliminated and not considered during vaccine design.

[0514] In some embodiments, the method further includes running the OptiVax-Robust algorithm as described in Liu et al. (2020a) using the HLA haplotype frequencies of a population on the scored candidate set to construct a base set (also referred to as seed set herein) of target peptides (FIG.2). In some embodiments, HLA diplotype frequencies can be provided to OptiVax. OptiVax-Robust includes algorithms to eliminate peptide redundancy that arises from the sliding window approach with varying window sizes, but other redundancy elimination measures can be used to enforce minimum edit distance constraints between target peptides in the candidate set. The size of the seed set is determined by a point of diminishing returns of population coverage as a function of the number of target peptides in the seed set. Other criteria can also be used, including a minimum number of vaccine target peptides, maximum number of vaccine target peptides, and desired predicted population coverage. In some embodiments, a predetermined population coverage is less than about 0.4, between about 0.4 and 0.5, between about 0.5 and 0.6, between about 0.6 and 0.7, between about 0.7 and 0.8, between about 0.8 and 0.9, or greater than about 0.9. Another possible criterion is a minimum number of expected peptide-HLA binding hits in each individual. In alternate embodiments, the method further includes running the OptiVax-Unlinked algorithm as described in Liu et al. (2020a) instead of OptiVax-Robust.

[0515] The OptiVax-Robust method uses binary predictions of peptide-HLA immunogenicity, and these binary predictions can be generated as described in Liu et al. (2020b). The OptiVax-Unlinked method uses the probability of target peptide binding to HLA - 84 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 alleles and can be generated as described in Liu et al. (2020a). In some embodiments, OptiVax- Unlinked and EvalVax-Unlinked are used with the probabilities of peptide-HLA immunogenicity. Either method can be used for the purposes described herein, and thus the term “OptiVax” refers to either the Robust or Unlinked method. In some embodiments, the observed probability of peptide-HLA immunogenicity in experimental assays can be used as the probability of peptide-HLA binding in EvalVax-Unlinked and OptiVax-Unlinked. In some embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design describe the world’s population. In alternative embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design are specific to a geographic region. In alternative embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design are specific to an ancestry. In alternative embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design are specific to a race. In alternative embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design are specific to individuals with risk factors such as genetic indicators of risk, age, exposure to chemicals, alcohol use, chronic inflammation, diet, hormones, immunosuppression, infectious agents, obesity, radiation, sunlight, or tobacco use. In alternative embodiments, the HLA haplotype or HLA allele frequencies of a population provided to OptiVax for vaccine design are specific to individuals that carry certain HLA alleles. In alternative embodiments, the HLA diplotypes provided to OptiVax for vaccine design describe a single individual, and are used to design an individualized vaccine.

[0516] In some embodiments, the base (or seed) set of target peptides (e.g., first peptide set) that results from OptiVax application to the candidate set of target peptides describes a set of unmodified target peptides that represent a possible compact vaccine design (Seed Set in FIG. 2). A base peptide is a target peptide that is included in the base or seed peptide set (e.g., first peptide set). In some embodiments, the seed set (e.g., first peptide set) is based upon filtering candidate peptide scores by predicted or observed affinity or immunogenicity with respect to HLA molecules (Seed Set in FIG.1). However, to improve the display of the target peptides in a wide range of HLA haplotypes as possible, some embodiments include modifications of the seed (or base) set. In some embodiments, experimental assays can be used to ensure that a modified seed (or base) peptide activates T cells that also recognize the base / seed peptide. - 85 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0517] For a given target peptide, the optimal anchor residue selection may depend upon the HLA allele that is binding to and displaying the target peptide and the class of the HLA allele (MHC class I or class II). A seed peptide set (e.g., first peptide set) can become an expanded set by including anchor residue modified peptides of either MHC class I or II peptides (FIGs.1-2). Thus, one aspect of vaccine design is considering how to select a limited set of heteroclitic peptides that derive from the same target peptide for vaccine inclusion given that different heteroclitic peptides will have different and potentially overlapping population coverages.

[0518] In some embodiments, all possible anchor modifications for each base set of target peptide are considered. There are typically two anchor residues in peptides bound by MHC class I molecules, typically at positions 2 and 9 for 9-mer peptides. In some embodiments, anchors for 8-mers, 10-mers, and 11-mers are found at positions 2 and n, where n is the last position (8, 10, and 11, respectively). For MHC class I molecules, the last position n is called the “C” position herein for carboxyl terminus. In some embodiments, at each anchor position, 20 possible amino acids are attempted in order to select the best heteroclitic peptides. Thus, for MHC class I binding, 400 (i.e., 20 amino acids by 2 positions = 202) minus 1 heteroclitic peptides are generated for each base target peptide. There are typically four anchor residues in peptides bound by MHC class II molecules, typically at positions 1, 4, 6, and 9 of the 9-mer binding core. Thus, for MHC class II binding there are 160,000 (i.e., 20 amino acids by 4 positions = 204) minus 1 heteroclitic peptides generated for each base target peptide. In some embodiments, more than two (MHC class I) or four (MHC class II) positions are considered as anchors. Other methods, including Bayesian optimization, can be used to select optimal anchor residues to create heteroclitic peptides from each seed (or base) set peptide. Other methods of selecting optimal anchor residues are presented in “Machine learning optimization of peptides for presentation by class II MHCs” by Dai et al. (2020), incorporated in its entirety herein. In some embodiments, the anchor positions are determined by the HLA allele that presents a peptide, and thus the set of heteroclitic peptides includes for each set of HLA specific anchor positions, all possible anchor modifications.

[0519] In some embodiments, for all of the target peptides in the base / seed set, new peptide sequences with all possible anchor residue modifications (e.g., MHC class I or class II) are created resulting in a new heteroclitic base set (Expanded set in FIGs.1-2) that includes all of the modifications. In some embodiments, anchor residue modifications of a peptide are not included in the heteroclitic base set if one or more of the peptide’s anchor residue positions - 86 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 contains a substitution mutation that distinguishes the peptide from a self-peptide. In some embodiments, anchor residue modifications of a base / seed peptide are only included in the heteroclitic base set for peptide positions that do not contain a substitution mutation that distinguishes the base / seed peptide from a self-peptide. In some embodiments, anchor residue modifications of a peptide are not included in the heteroclitic base set when one or more of the peptide’s mutations does not occur between a pair of its adjacent anchor residues. In some embodiments, for all of the target peptides in the base / seed set, new peptide sequences with anchor residue modifications (e.g., MHC class I or class II) at selected anchor locations are created resulting in a new heteroclitic base set (Expanded set in FIGs.1-2) that includes the selected modifications. In some embodiments, the anchor residue positions used for modifying peptides are selected from anchor residue positions determined by the HLA alleles considered during vaccine evaluation. In some embodiments, the heteroclitic base set (Expanded set in FIGs.1-2) also includes the original seed (or base) set (Seed Peptide Set in FIGs.1-2). In some embodiments, the heteroclitic base set includes amino acid substitutions at non-anchor residues. In some embodiments, modifications of base peptide residues is accomplished to alter binding to T cell receptors to improve therapeutic efficacy (Candia, et al.2016). In some embodiments, the heteroclitic base set includes amino acid substitutions of non-natural amino acid analogs. The heteroclitic base set is scored for HLA affinity, peptide-HLA immunogenicity, or other metrics as described herein (another round of Peptide Scoring and Score Filtering as shown in FIGs.1- 2).

[0520] In some embodiments, the scoring predictions may be further updated for pairs of heteroclitic peptide and HLA allele, eliminating pairs where a heteroclitic peptide has a seed (or base) peptide from which it was derived that is not predicted to be displayed by the HLA allele at a specified threshold of peptide-HLA binding score or a specified peptide-HLA immunogenicity metric. In some embodiments, at the second Peptide Scoring and Score Filtering step in FIGs.1 and 2 a peptide-HLA score is not used for a heteroclitic peptide for a given HLA for vaccine design when the product (or other function) of the heteroclitic peptide’s peptide-HLA immunogenicity or binding probability score and the peptide-HLA immunogenicity or binding probability score for its unmodified counterpart peptide do not meet a specified threshold (e.g., 0.5, 0.6, 0.7, 0.8, or 0.9). In some embodiments, the peptide-HLA scores may also be filtered to ensure that predicted binding cores of the heteroclitic peptide displayed by a particular HLA allele align exactly in position with the binding cores of the respective seed (or base) set target peptide for that HLA allele. In some embodiments, the - 87 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 scoring predictions are filtered for an HLA allele to ensure that the heteroclitic peptides considered for that HLA allele are only modified at anchor positions determined by that HLA allele. Scoring produces a metric of peptide-HLA immunogenicity for peptides and HLA alleles that can be either binary, a probability of immunogenicity, or other metric of immunogenicity such as peptide-HLA affinity or percent rank, and can be based on computational predictions, experimental observations, or a combination of both computational predictions and experimental observations.

[0521] In some embodiments, probabilities of peptide-HLA immunogenicity are utilized by OptiVax-Unlinked. In some embodiments, heteroclitic peptides are included in experimental assays such as MIRA (Klinger et al., 2015) or ELISPOT to determine their peptide-HLA immunogenicity metric with respect to specific HLA alleles. In some embodiments, the methods of Liu et al. (2020b), can be used to incorporate MIRA data for heteroclitic peptides into a model of peptide-HLA immunogenicity. In some embodiments, peptide-HLA immunogenicity metrics of heteroclitic peptides are experimentally determined and their ability to activate T cells that also recognize the corresponding seed (or base) peptide of the heteroclitic peptide is performed as is known in the art to qualify the heteroclitic peptide for vaccine inclusion (e.g., Houghton et al., 2007). In some embodiments, these assays of the immunogenicity and cross-reactivity of heteroclitic peptides are performed when the heteroclitic peptides are displayed by specific HLA alleles.

[0522] In some embodiments, experimental observations of the display of heteroclitic peptides by specific HLA alleles in cells can be used to score peptides for peptide-HLA binding or peptide-HLA immunogenicity. In some embodiments, mass spectrometry is used to experimentally determine the display of heteroclitic peptides by cells as described by Bear et al. (2021) or Wang et al. (2019) and these data are used to score for peptide-HLA binding or peptide-HLA immunogenicity. In some embodiments, mass spectrometry is used to experimentally determine the display of heteroclitic peptides by cells, and these experimental data are used to qualify the inclusion of heteroclitic peptides for inclusion in a vaccine. In some embodiments, mass spectrometry is used to experimentally determine the display of a peptide by tumor cells, and these experimental data are used to exclude peptide-HLA binding scores or peptide-HLA immunogenicity scores for the peptide when the peptide is not observed to be displayed by an HLA allele by mass spectrometry. In some embodiments, mass spectrometry is used to experimentally determine the display of a heteroclitic peptide by cells with an HLA - 88 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 allele found in an individual, and these experimental data are used to qualify the inclusion of the heteroclitic peptide for inclusion in a vaccine for the individual. In some embodiments, mass spectrometry is used to experimentally determine the display of a peptide by tumor cells in an individual, and these experimental data are used to exclude peptide-HLA binding scores or peptide-HLA immunogenicity scores for the peptide when the peptide is not observed to be displayed by an HLA allele by mass spectrometry. In some embodiments, computational predictions of the immunogenicity of a heteroclitic peptide in the context of display by HLA alleles can used for scoring such as the methods of Ogishi et al. (2019) or Bulik-Sullivan et al. (2019).

[0523] In some embodiments, a peptide in the heteroclitic base set is removed if (1) one of its anchor positions for an HLA allele corresponds to the location of a mutation in the base / seed peptide from which it was derived that distinguishes the base / seed peptide from a self-peptide, and (2) if the peptide-HLA binding or peptide-HLA immunogenicity of the self-peptide is stronger than a specified threshold for self-peptide binding or immunogenicity. This eliminates peptides in the heteroclitic base set that may cross-react with self-peptides as a result of sharing TCR facing residues with self-peptides. In some embodiments, the threshold for self-peptide binding is between approximately 500 nM to 1000 nM.

[0524] In some embodiments, redundant peptides in the heteroclitic base set are removed. In some embodiments, a redundant peptide is a first heteroclitic peptide that has peptide-HLA immunogenicity scores or peptide-HLA binding scores that are less immunogenic for all scored HLAs than a second heteroclitic peptide in the heteroclitic base set, where both the first and second heteroclitic peptides are derived from the same base (or seed) peptide. In some embodiments, peptide redundancy is determined by only comparing peptide-HLA immunogenicity scores or peptide-HLA binding scores for HLA alleles where the peptide-HLA immunogenicity scores or peptide-HLA binding scores for both peptides for an HLA allele are more immunogenic than a given threshold (e.g., 50 nM for binding). In some embodiments, a redundant peptide is a first heteroclitic peptide that has an average peptide-HLA immunogenicity score or peptide-HLA binding score that is less immunogenic than the average peptide-HLA immunogenicity score or peptide-HLA binding score of a second heteroclitic peptide in the heteroclitic base set, where both the first and second heteroclitic peptides are derived from the same base (or seed) peptide, and the average scores are computed for HLA alleles where the peptide-HLA immunogenicity scores or peptide-HLA binding scores for both - 89 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 peptides for an HLA allele are more immunogenic than a given threshold (e.g., 50 nM for binding). In some embodiments, a redundant peptide is a first heteroclitic peptide that has a weighted peptide-HLA immunogenicity score or peptide-HLA binding score that is less immunogenic than the weighted peptide-HLA immunogenicity score or peptide-HLA binding score of a second heteroclitic peptide in the heteroclitic base set, where both the first and second heteroclitic peptides are derived from the same base (or seed) peptide, and where the weighting is determined by the frequency of the HLA allele in a human population, and the weighted scores are computed for HLA alleles where the peptide-HLA immunogenicity scores or peptide- HLA binding scores for both peptides for an HLA allele are more immunogenic that a given threshold (e.g., 50 nM for binding).

[0525] In some embodiments, the next step involves scoring the heteroclitic base set (the second peptide set) and filtering the resulting scores to create a second peptide set by comparing the peptide-HLA immunogenicity scores or peptide-HLA binding scores of the peptides for one or more HLA alleles to a threshold. In some embodiments, an affinity criterion of about 50 nM is used to increase the probability that a vaccine peptide will be found and displayed by HLA molecules. In some embodiments, the affinity criteria is more constrained than 50 nM (i.e., < 50 nM). In some embodiments, the affinity criteria is more constrained than about 500 nM (i.e., < 500 nM). In some embodiments, individual peptide-HLA binding scores or immunogenicity metrics are determined and thus a peptide may be retained as long as it meets the criteria for at least one HLA allele, and only peptide-HLA scores that meet the criteria are considered for vaccine design.

[0526] In some embodiments, probabilistic thresholds are used in the peptide scoring and score filtering steps (FIGs.1-2) to filter peptide-HLA combinations for vaccine design. In some embodiments, a credence function is used to predict the probability of peptide-HLA display or peptide-HLA immunogenicity for each peptide for a desired set of HLA alleles. In some embodiments, a credence function implementation fits the output of a binding prediction algorithm (such the EL or BA output of NetMHCpan4.1 or NetMHCIIpan4.0) to observed held- out binding or immunogenicity data. One implementation of credence functions is described in Dai, Z., & Gifford, D. “Constrained Submodular Optimization for Vaccine Design”. arXiv preprint arXiv:2206.08336. https: / / arxiv.org / abs / 2206.08336, Version 2, 27 January 2023. Held-out data is experimental data that is not used to train the binding prediction algorithm. In some embodiments, held-out data contains examples of experimentally verified peptide-HLA - 90 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 binding or immunogenicity. In some embodiments, held-out data describes known peptide-HLA binding pairs, and peptides surrounding known binders in these held-out data are used to generate negative examples of binding which is added to the held-out data. The credence function that maps the output of a prediction algorithm to a probability of binding (or immunogenicity) is selected to minimize the difference between the output of the prediction algorithm on peptides examples in the held-out data and the experimentally observed binding (or immunogenicity) in the held-out data. For example, when the held-out data contains an experimentally verified peptide-HLA binding pair, the output of the credence function should assign this peptide-HLA pair a high probability of binding. In some embodiments, this is accomplished by dividing the output of the prediction algorithm into discrete intervals (“bins”) and for each bin choosing a function that maps the output of the prediction algorithm that are contained in that bin into the observed fraction of binding (or immunogenicity) in the held-out experimental data. In some embodiments, the functions for each interval are chosen so that increasing intervals have increasing probabilities of binding (or immunogenicity). In some embodiments, the process of fitting a credence function is repeated on different sets of held-out data, and the difference between the credence functions on each set of held-out data is used to validate the accuracy of the credence function.

[0527] In some embodiments, the first peptide scoring and filtering step uses a credence function to predict the probability of binding (or immunogenicity) for all combinations of candidate peptides and HLA alleles. In some embodiments, the first peptide scoring and filtering step eliminates peptide-HLA combinations that do not bind (or are not immunogenic) stronger than a probability threshold for the respective HLA allele. In some embodiments, the second peptide scoring and filtering step eliminates peptide-HLA combinations where (1) the peptide’s corresponding base peptide-HLA combination was eliminated in the first peptide scoring and filtering step, or (2) the peptide-HLA combination does not have a probability greater than a second more stringent probability threshold. In some embodiments, the second peptide scoring and filtering step uses a function to combine the peptide-HLA display (or immunogenicity) probability of a base peptide with the peptide-HLA display (or immunogenicity) probability of its heteroclitic derivative, and the result of the function must be greater than a threshold to keep the peptide-HLA combination for the heteroclitic derivative. In some embodiments, the combination function is multiplication. In some embodiments, the result of the combination function is used as the peptide-HLA metric for that peptide for vaccine design. - 91 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0528] In some embodiments, the next step involves inputting the second peptide set to OptiVax to select a compact set of vaccine peptides that maximizes predicted vaccine performance (Vaccine Performance Optimization; FIGs.1-2). In some embodiments, predicted vaccine performance is a function of expected peptide-HLA binding affinity (e.g., a function of the distribution of peptide-HLA binding affinities across all peptide-HLA combinations for a given peptide set, or weighted by the occurrence of the HLA alleles in a population or individual). In some embodiments, predicted vaccine performance is the expected population coverage of a vaccine. In some embodiments, predicted vaccine performance is the expected number peptide-HLA hits produced by a vaccine in a population or individual. In some embodiments, predicted vaccine performance requires a minimum expected number of peptide- HLA hits (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) produced by a vaccine. In some embodiments, predicted vaccine performance is a function of population coverage and expected number of peptide-HLA hits desired produced by a vaccine. In some embodiments, predicted vaccine performance is a metric that describes the overall immunogenic properties of a vaccine where all of the peptides in the vaccine are scored for peptide-HLA immunogenicity for two or more HLA alleles (e.g., three or more HLA alleles). In some embodiments, predicted vaccine performance excludes immunogenicity contributions by selected HLA alleles above a maximum number of peptide-HLA hits (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more). In some embodiments, predicted vaccine performance excludes immunogenicity contributions of individual HLA diplotypes above a maximum number of peptide-HLA hits (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more). In some embodiments, predicted vaccine performance is the fraction of covered HLA alleles, which is the expected fraction of HLA alleles in each individual that have a minimum number of peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) with predicted peptide-HLA immunogenicity produced by a vaccine. In some embodiments, predicted vaccine performance is the expected fraction of HLA alleles in a single individual that have a minimum number of peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) with predicted peptide-HLA immunogenicity produced by a vaccine.

[0529] In some embodiments, a vaccine is designed by the iterative selection of peptides from the heteroclitic base set (also referred to as Expanded set as shown in FIGs.1-2) at progressively less stringent criteria for predicted peptide immunogenicity or display. In some embodiments, a peptide is retained if at least one of its peptide-HLA scores is not eliminated by the thresholds employed. In some embodiments, OptiVax is first used to design a vaccine with a desired vaccine performance with specific peptide qualification criteria (e.g., seed HLA-peptide scores from the candidate set must bind to at least one MHC molecule at 500 nM or stronger, - 92 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 and peptide-HLA scores from the expanded set must bind to at least one MHC molecule at 50 nM or stronger). The vaccine that results from this application of OptiVax is then used as the foundation for vaccine augmentation with less stringent criteria (e.g., seed peptide-HLA scores from the candidate set must bind to at least one MHC molecule at 1000 nM or stronger, and peptide HLA-scores from the expanded set must bind to at least one MHC molecule at 100 nM or stronger) to further improve the desired vaccine performance. Methods for vaccine augmentation are described in Liu et al. (2020b), incorporated by reference in its entirety herein. In some embodiments, multiple rounds of vaccine augmentation may be utilized. In some embodiments, the final augmented vaccine is the one selected.

[0530] In some embodiments, selection of peptide sets to meet a desired predicted vaccine performance can be accomplished by computational algorithms other than OptiVax. In some embodiments, integer linear programming or mixed-integer linear programming is employed for selecting peptide sets instead of OptiVax. One example of an integer programming method for peptide set selection is described by Toussaint et al., 2008, incorporated by reference in its entirety herein. An example solver for mixed-integer linear programming is Python-MIP that can be used in conjunction with Toussaint et al., 2008. A second example of methods for vaccine peptide selection is described in “Maximum n-times Coverage for Vaccine Design” by Liu et al. (2021), incorporated by reference in its entirety herein.

[0531] Predicted vaccine performance refers to a metric. Predicted vaccine performance can be expressed as a single numerical value, a plurality of numerical values, any number of non- numerical values, and a combination thereof. The value or values can be expressed in any mathematical or symbolic term and on any scale (e.g., nominal scale, ordinal scale, interval scale, or ratio scale).

[0532] A seed (or base) peptide and all of the modified peptides that are derived from that seed (or base) peptide comprise a single peptide family. In some embodiments, in the component of vaccine performance that is based on peptide-HLA immunogenicity for a given HLA allele, a maximum number of peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) that are in the same peptide family are given computational immunogenicity credit for that HLA allele. This limit on peptide family immunogenicity limits the credit caused by many modified versions of the same base peptide. In some embodiments, the methods described herein are included for running OptiVax with an EvalVax objective function that corresponds to a desired metric of predicted vaccine performance. In some embodiments, population coverage means the - 93 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 proportion of a subject population that presents one or more immunogenic peptides that activate T cells responsive to a seed (or base) target peptide. The metric of population coverage is computed using the HLA haplotype frequency in a given population such as a representative human population. In some embodiments, the metric of population coverage is computed using marginal HLA frequencies in a population. Maximizing population coverage means selecting a peptide set (either a base peptide set, a modified peptide set, or a combination of base and modified peptides; e.g., a first peptide set, second peptide set, or third peptide set) that collectively results in the greatest fraction of the population that has at least a minimum number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) of immunogenic peptide-HLA bindings based on proportions of HLA haplotypes in a given population (e.g., representative human population). In some embodiments, this process includes the OptiVax selection of heteroclitic peptides (as described in this disclosure) that activate T cells that respond to their corresponding seed (or base) peptide and the heteroclitic base peptides to improve population coverage. In some embodiments, the seed (or base) target peptides are always included in the final vaccine design. In some embodiments, peptides are only considered as candidates for a vaccine design (e.g., included in a first, second, and / or third peptide set) if they have been observed to be immunogenic in clinical data, animal models, or tissue culture models. In some embodiments, vaccine peptides are selected to be displayed by a peptide specific set of HLA class I or class II alleles, wherein for at least two peptides in a vaccine all of the peptide specific sets of HLA class I or class II alleles are not identical.

[0533] Although heteroclitic peptides are used as exemplary embodiments in this disclosure, any modified peptide could be used in place of a heteroclitic peptide. A modified peptide is a peptide that has one or more amino acid substitutions of a target base / seed peptide. The amino acid substitution could be located at an anchor position or any other non-anchor position.

[0534] In some embodiments, a candidate vaccine peptide (e.g., a base peptide or a modified peptide) is eliminated from vaccine inclusion if it activates T cells that recognize self-peptides (e.g., this can be achieved at the first and / or second round of Peptide Filtering and Sorting as shown in FIGs.1-2). In some embodiments, a candidate vaccine peptide (e.g., a base peptide or a modified peptide) is computationally eliminated from vaccine inclusion if its outward facing amino acids, when bound by an HLA allele, are similar to outward facing self-peptide residues that are presented by the same HLA allele, where similarity can be defined by identity or similarity metrics such as BLOSUM matrices (BLOSUM matrices are known in the art). In - 94 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 some embodiments, calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two protein sequences can be determined by aligning the proteins and then computing the percentage of residues that are identical as described in Wang et al. (BMC Bioinformatics 19, 529 (2018).

[0535] Testing a vaccine peptide for its ability to activate T cells that recognize self-peptides can be experimentally accomplished by the vaccination of animal models followed by ELISPOT or other immunogenicity assay or with human tissue protocols. In both cases, models with HLA alleles that present the vaccine peptide are used. In some embodiments, human primary blood mononuclear cells (PBMCs) are stimulated with a vaccine peptide, the T cells are allowed to grow, and then T cell activation with a self-peptide is assayed as described in Tapia-Calle et al. (2019) or other methods as known in the art. In some embodiments, the vaccine peptide is excluded from vaccine inclusion if the T cells are activated by the self-peptide. In some embodiments, computational predictions of the ability of a peptide to activate T cells that also recognize self-peptides can be utilized. These predictions can be based upon the modeling of the outward facing residues from the peptide-HLA complex and their interactions with other peptide residues. In some embodiments, a candidate vaccine peptide (e.g., a base peptide or a modified peptide) is eliminated from vaccine inclusion or experimentally tested for cross-reactivity if it is predicted to activate T cells that also recognize self-peptides based upon the structural similarity of the peptide-MHC complex of the candidate peptide (e.g., a base peptide or a modified peptide) and the peptide-MHC complex of a self-peptide. One method for the prediction of peptide-MHC structure is described by Park et al. (2013).

[0536] In some embodiments, the peptide-HLA binding score or peptide-HLA immunogenicity metric for a candidate heteroclitic vaccine peptide (e.g., a modified peptide) and HLA allele is eliminated from consideration during vaccine design if the candidate heteroclitic vaccine peptide does not activate T cells that recognize its corresponding base / seed target peptide (second round of Peptide Scoring and Score Filtering, FIGs.1-2) for the given HLA allele. In some embodiments, a heteroclitic vaccine peptide (e.g., a modified peptide) is - 95 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 eliminated from a vaccine design if the candidate heteroclitic vaccine peptide does not activate T cells that recognize its corresponding base / seed target peptide (second round of Peptide Scoring and Score Filtering, FIGs.1-2) for a given HLA allele. Testing a candidate heteroclitic peptide (e.g., a modified peptide) for its ability to activate T cells that recognize its corresponding seed (or base) target peptide with respect to the same HLA allele can be experimentally accomplished by the vaccination of animal models followed by ELISPOT or other immunogenicity assay or with human tissue protocols. In both cases, models with HLA alleles that present the heteroclitic peptide are used. In some embodiments, human PBMCs are stimulated with the heteroclitic peptide, the T cells are allowed to grow, and then T cell activation with the seed (or base) target peptide is assayed as described in Tapia-Calle et al. (2019) or using other methods known in the art. In some embodiments, computational predictions of the ability of a heteroclitic peptide to activate T cells that also recognize the corresponding seed (or base) target peptide can be utilized. These predictions can be based upon the modeling of the outward facing residues from the peptide-HLA complex and their interactions with other peptide residues. In some embodiments, the structural similarity of the peptide-HLA complex of a heteroclitic peptide and the peptide-HLA complex of the corresponding seed (or base) target is used to qualify heteroclitic peptides for vaccine inclusion or to require experimental immunogenicity testing before vaccine inclusion.

[0537] TCR Interface Divergence (TCRID) is the Least Root Mean Square Deviation of the difference between a first peptide’s TCR facing residues’ 3D positions and the corresponding residue positions of a second peptide with respect to a specific HLA allele. In some embodiments, other metrics are used for the TCRID instead of Least Root Mean Square Deviation. In some embodiments, other metrics are used for the TCRID that include position deviations in non-TCR facing residues and MHC residues from the specific HLA allele. In some embodiments, TCRID is used to predict if two peptides when displayed by a given HLA allele will activate the same T cell clonotypes. In some embodiments, FlexPepDock (London et al., 2011, incorporated by reference in its entirety herein) or DINC (Antunes et al., 2018, incorporated by reference in its entirety herein) in conjunction with the crystal structures of HLA molecules can be used to compute TCRID metrics for pairs of peptides given an HLA molecule. In some embodiments, TCRID is computed by (1) determining the 3D peptide-HLA structures for two different peptides bound by a specific HLA allele, (2) aligning the HLA alpha helices of the peptide-HLA structures, and (3) computing the Least Root Mean Square Deviation of the - 96 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 difference between the TCR facing residues of the two peptides with respect to the aligned alpha helix reference frame.

[0538] In some embodiments, the second Peptide Scoring and Score Filtering step in FIGs.1 and 2 will eliminate the peptide-HLA binding or immunogenicity score for a heteroclitic peptide for a specific HLA allele when the HLA specific TCRID between the heteroclitic peptide and its corresponding base (or seed) peptide from which it was derived is over a first TCRID threshold. In some embodiments, the second Peptide Scoring and Score Filtering step in FIGs.1 and 2 will eliminate all peptide-HLA binding or immunogenicity scores for a heteroclitic peptide when the HLA specific TCRID between the heteroclitic peptide and its corresponding unmutated self- peptide from which it was derived is under a second TCRID threshold. In some embodiments, the first Peptide Scoring and Score Filtering step in FIGs.1 and 2 will eliminate all peptide-HLA binding or immunogenicity scores for a candidate peptide when the HLA specific TCRID between the peptide and its corresponding unmutated self-peptide is under a third TCRID threshold. In some embodiments, any of the TCRID thresholds are determined by experimentally observing or computationally predicting the cross-reactivity of TCR molecules to peptide-HLA complexes.

[0539] FIG.3 (MHC class I) and FIG.4 (MHC class II) show the predicted population coverage of OptiVax-Robust selected single target-specific vaccines with differing number of peptides designed for selected mutations in the BRAF, CTNNB1, KRAS, PIK3CA, and TP53 genes. FIGs.3-4 show that as the number of peptides increases for a vaccine, its predicted population coverage increases. The population coverage shown in FIGs.3-4 are of those individuals that have the specific mutation that the vaccine is designed to cover. An increase in peptide count will also typically cause the average number of peptide-HLA hits in each individual to increase in the population.

[0540] OptiVax can be used to design a vaccine to maximize the fraction / proportion of the population whose HLA molecules are predicted to bind to and display at least p peptides from the vaccine. In some embodiments, this prediction (e.g., scoring) includes experimental immunogenicity data to directly predict at least p peptides will be immunogenic. The number p is input to OptiVax, and OptiVax can be run multiple times with varying values for p to obtain a predicted optimal target peptide set for different peptide counts p. Larger values of p will increase the redundancy of a vaccine at the cost of more peptides to achieve a desired population coverage. In some embodiments, it may not be possible to achieve a given population coverage - 97 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 given a specific heteroclitic base set. In some embodiments, the number p is a function of the desired size of a vaccine.

[0541] The methods described herein can be used to design separate vaccine formulations for MHC class I and class II-based immunity.

[0542] In some embodiments, this procedure is used to create a vaccine for an individual. In some embodiments, the target peptides present in the individual are determined by sequencing the individual’s tumor RNA or DNA, and identifying mutations that produce foreign peptides. One embodiment of this method is described in U.S. Patent No.10,738,355, incorporated by reference in its entirety herein. In some embodiments, peptide sequencing methods are used to identify target peptides in the individual. One embodiment of this is described in U.S. Patent Publication No.2011 / 0257890. In some embodiments, the target peptides used for the individual’s vaccine are selected when a self-peptide, foreign peptide, pathogen peptide or RNA encoding a self-peptide, foreign peptide, or pathogen peptide observed in a specimen from the individual is present at a predetermined level. The target peptides in the individual are used to construct a vaccine as disclosed herein. For vaccine design, OptiVax is provided a diplotype comprising the HLA type of the individual. In an alternative embodiment, the HLA type of an individual is separated into multiple diplotypes with frequencies that sum to one, where each diplotype comprises one or more HLA alleles from the individual and a notation that the other allele positions should not be evaluated. The use of multiple diplotypes will cause OptiVax’s objective function to increase the chance that immunogenic peptides will be displayed by all of the constructed diplotypes. This achieves the objective of maximizing the number of distinct HLA alleles in the individual that exhibit peptide-HLA immunogenicity and thus improves the allelic coverage of the vaccine in the individual.

[0543] FIG.5 shows the predicted vaccine performance (predicted number of peptide-HLA hits) of ten example G12V MHC class I vaccines for a single individual with the MHC class I HLA diplotype HLA-A02:03, HLA-A11:01, HLA-B55:02, HLA-B58:01, HLA-C03:02, HLA- C03:03. OptiVax was used to design ten G12V MHC class I vaccines for this HLA diplotype with peptide counts ranging from 1 to 10. For the results in FIG.5, OptiVax was run with six synthetic diplotypes, each equally weighted, each with one HLA allele from the individual’s HLA diplotype, and the other allele positions marked to not be evaluated. - 98 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 MHC Class I Vaccine Design Procedure

[0544] In some embodiments, MHC class I vaccine design procedures consist of the following computational steps.

[0545] In some embodiments, the inputs for the computation are: sequence (length n) containing the neoantigen(s) or pathogenic target(s) of interest (e.g., KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, KRAS G13D). ^^^denotes the amino acid at position i. t: Position of target mutation in ^^, ^^ ∈^1, … ^^^(e.g., t = 12 for KRAS G12D). s: Substitution mutation ^^ ∈ ^ ^^ ^^ ^^ ^^, ^^ ^^ ^^ ^^ ^^^ is true if the mutation is a substitution, and false if the mutation is a deletion or insertion or the peptide does not contain a mutation (such as in pathogen targets). When the mutation is a deletion or insertion then t indicates the position immediately before the deletion or insertion. ^^^: Threshold for potential presentation of peptides by MHC for peptide-MHC scoring (e.g., 500 nM binding affinity) ^^ଶ: Threshold for predicted display of peptides by MHC for peptide-MHC scoring (e.g., 50 nM binding affinity) ℋ: Set of HLA alleles (for HLA-A, HLA-B, HLA-C loci) F: ℝ: Population haplotype frequencies (for OptiVax optimization and coverage evaluation). N: Parameter for EvalVax and OptiVax objective function. Specifies minimum number of predicted per-individual hits for population coverage objective to consider the individual covered. Default = 1 (computes P(n ≥ 1) population coverage).

[0546] In some embodiments, Peptide-HLA Scoring Functions used are: ScorePotential : ^^ ×ℝ: Scoring function mapping a (peptide, HLA allele) pair to a prediction of peptide-HLA display. If predicted affinity ≤ τ1, then returns 1, else returns 0. Options include MHCflurry, NetMHCpan, PUFFIN, ensembles, or alternative metrics or software may be used, including models calibrated against immunogenicity data. SCOREDISPLAY : ^^ × ℋ → ℝ: Scoring function mapping a (peptide, HLA allele) pair to a prediction of peptide-HLA display. If predicted affinity ≤ τ2, then returns 1, else returns 0. - 99 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Options include MHCflurry, NetMHCpan, PUFFIN, ensembles, or alternative metrics or software may be used, including models calibrated against immunogenicity data.

[0547] Next, from the seed protein sequence (P), a set ^^ of windowed native peptides spanning the protein sequence(s) is constructed. ^^^…^ା(^ି^)only produces set members when the subscripts are within the range of the defined seed protein P. In some embodiments, 8-mers, 9-mers, 10-mers, and 11-mers are produced, but this process can be performed with any desired window lengths and the resulting peptide sets combined. In some embodiments, only 9-mers are produced. ^^ = ^ ^^^^∈^଼,…,^^^^^^^1 ^

[0548] The second condition j ≠ {t – (k-1), t – 1} excludes peptides where the mutation at t is in positions P2 or Pk of the windowed k-mer peptide (i.e., the anchor positions) and the mutation is a substitution. MHC Class I Vaccine Design Procedure with Defined Peptide Set ^^

[0549] Next, each peptide sequence in ^^ is scored against all HLA alleles in ℋ for potentialpresentation using SCOREPOTENTIAL (with threshold τ1 = 500 nM) and store results in a| ^^| × |ℋ| matrix S:S[p, h] = SCOREPOTENTIAL(p, h) ∀ p ∈ ^^, h ∈ ℋ Note that S is a binary matrix where 1 indicates the HLA is predicted to potentially present the peptide, and 0 indicates no potential presentation. Define base set of peptides ^^ ⊆ ^^: ^^ = ^ ^^ ∈ ^^ | ∃ℎ s. t. ^^^ ^^,ℎ^ = 1^ Thus, B contains the native peptides that are predicted to be potentially presented by at least 1 HLA. - 100 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Create a set of all heteroclitic peptides B' stemming from peptides in B:where ANCHOR-MODIFIED(b) returns a set of all 399 anchor-modified peptides stemming from b (with all possible modifications to the amino acids at P2 and P9).

[0550] Next, all heteroclitic candidate peptides (e.g., modified peptides) in B' are scored against all HLA alleles in ℋ for predicted display using SCOREDISPLAY (with threshold τ2= 50 nM), and store results in binary |B'| × |ℋ| matrix ^^^ᇱ: ^^^ᇱ^ ^^ᇱ,ℎ^ = SCOREDISPLAY( ^^ᇱ,ℎ) ∀ ^^ᇱ∈ ^^ᇱ,ℎ ∈ ℋ

[0551] Next, an updated scoring matrix ^^ଶᇱis computed for heteroclitic peptides conditioned on the potential presentation of the corresponding base peptides by each HLA: ^^ᇱ^^ ^^ᇱ, ℎ^, if ^^^ ^^,ℎ^ = 10, otherwise ∀ ^^ᇱ∈ ^^ᇱ,ℎ ∈ ℋwhere each heteroclitic peptide b' ∈ B' is a mutation of base peptide b ∈ B. This condition enforces that if h was not predicted to potentially present b, then all heteroclitic peptides b' derived from b will not be displayed by h (even if h would otherwise be predicted to display b').

[0552] In some embodiments, OptiVax-Robust is used to design a final peptide set (e.g., third peptide set) from the union of base peptides and heteroclitic peptides B ∪ B' (with corresponding scoring matrices S and ^^ଶᇱfor B and B', respectively). OptiVax will output m sets ^^^for s ∈ [1, …, m] where m is the largest vaccine size requested from OptiVax. Let ^^^denote the compact set of vaccine peptides output by OptiVax containing k peptides. Note that ^^^ା^is not necessarily a superset of ^^^. In alternate embodiments, OptiVax can be used to augment the base set B with peptides from B' using scoring matrix ^^ଶᇱto have OptiVax return set ^^^, and the final vaccine setof peptides ^^ ∪ ^^^.

[0553] In some embodiments, this procedure is repeated independently for each target of interest, and the resulting independent vaccine sets can be merged into a combined vaccine as described below. - 101 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 MHC Class II Vaccine Design Procedure

[0554] In some embodiments, MHC class II vaccine design procedures consist of the following computational steps.

[0555] In some embodiments, the inputs for the computation are: sequence(s) (length n) containing the neoantigen(s) or pathogenic target(s) of interest (e.g., KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, KRAS G13D). Pi denotes the amino acid at position i. t: Position of target mutation in P, t ∈ [1,…,n] (e.g., t = 12 for KRAS G12D). s: Substitution mutation ^^ ∈^^^ ^^ ^^ ^^, ^^ ^^ ^^ ^^ ^^^is true if the mutation is a substitution, and false if the mutation is a deletion or insertion or the peptide does not contain a mutation (such as for pathogen targets). When the mutation is a deletion or insertion then t indicates the position immediately before the deletion or insertion. ^^^: Threshold for potential presentation of peptides by MHC for peptide-MHC scoring (e.g., 500 nM binding affinity) ^^ଶ: Threshold for predicted display of peptides by MHC for peptide-MHC scoring (e.g., 50 nM binding affinity) ℋ: Set of HLA alleles (for HLA-DR, HLA-DQ, HLA-DP loci) F : ℋଷ→ ℝ: Population haplotype frequencies (for OptiVax optimization and coverage evaluation). N: Parameter for EvalVax and OptiVax objective function. Specifies minimum number of predicted per-individual hits for population coverage objective to consider the individual covered. Default = 1 (computes P(n ≥ 1) population coverage).

[0556] In some embodiments, Peptide-HLA Scoring Functions used are: SCOREPOTENTIAL : ^^ × ℋ → ℝ: Scoring function mapping a (peptide, HLA allele) pair to a prediction of display. If predicted affinity ≤ τ1, then returns 1, else returns 0. Options include NetMHCIIpan, PUFFIN, ensembles, or alternative metrics or software may be used, including models calibrated against immunogenicity data. SCOREDISPLAY : ^^ × ℋ → ℝ: Scoring function mapping a (peptide, HLA allele) pair to a prediction of peptide-HLA display. If predicted affinity ≤ τ2, then returns 1, else returns 0. - 102 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 Options include NetMHCIIpan, PUFFIN, ensembles, or alternative metrics or software may be used, including models calibrated against immunogenicity data. FindCore : ^^ × ℋ → [1, ... , ^^]: Function mapping a (peptide, HLA allele) pair to a prediction of the 9-mer binding core. The core may be specified as the offset position (index) into the peptide where the core begins.

[0557] Next, from the seed protein sequence (P), a set ^^ of peptides spanning the protein sequence are constructed.only produces set members when the subscripts are within the range of the defined seed protein P. Here, we extract all windowed peptides of length 13–25 spanning the target mutation, but this process can be performed using any desired window lengths (e.g., only 15-mers).^where ^^^contains all sliding windows of length k, which are combined to form ^^. Note that here (unlike MHC class I), no peptides are excluded based on binding core or anchor residue positions (for MHC class II, filtering is performed as described in this disclosure). MHC Class II Vaccine Design Procedure with Defined Peptide Set ^^

[0558] Next, each peptide sequence in ^^ is scored against all HLA alleles in ℋ for potential presentation using SCOREPOTENTIAL (with threshold τ1 = 500 nM) and store results in a | ^^| × | ℋ| matrix S1: S1[p, h] = SCOREPOTENTIAL(p, h) ∀ p ∈ ^^, h ∈ ℋ Note that S1 is a binary matrix where 1 indicates the HLA is predicted to potentially present the peptide, and 0 indicates no potential presentation.

[0559] For each (peptide, HLA allele) pair (p, h), identify / predict the 9-mer binding core using FINDCORE. The predicted binding core is recorded in a matrix C: C[p, h] = FINDCORE(p, h) ∀ p ∈ ^^, h ∈ ℋ

[0560] Next, if not(s) then S2[p, h] = S1[p, h] otherwise an updated scoring matrix S2 is computed for native peptides in ^^: - 103 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 ^^ଶ[ ^^, ℎ] = ^^^^[ ^^, ℎ], if ^^[ ^^, ℎ] specifies ^^௧ at a non − anchor position inside0, otherwiseℋwhere Ptis the target residue of interest (e.g., the mutation site of KRAS G12D). This condition enforces the target residue to fall within the binding core at a non-anchor position for all (peptide, HLA allele) pairs with non-zero scores in S2 and allows the binding core to vary by allele per peptide (as the binding cores of a particular peptide may differ based on the HLA allele presenting the peptide). Thus, for each pair (p, h), if the predicted binding core C[p, h] specifies the target residue Pt at an anchor position (P1, P4, P6, or P9 of the 9-mer core), or if Ptis not contained within the binding core, then S2[p, h] = 0. In an alternate embodiment, Ptcan be located outside of the core or inside the core in a non-anchor position. In some embodiments, Pt can only be located at specific positions inside and / or outside of the core. In some embodiments, the binding core predictions in C are accompanied by prediction confidences. In some embodiments, if the confidence for predicted core C[p, h] is below a desired threshold (e.g., 0.5, 0.6, 0.7, 0.8, or 0.9), then S2 [p, h] = 0.

[0561] Next, OptiVax-Robust is run with peptides ^^ and scoring matrix S2 to identify a non- redundant base set of peptides B ⊆ ^^. (In alternate embodiments, B can be chosen as the entire set ^^ rather than identifying a non-redundant base set.)

[0562] Next, a set of all heteroclitic peptides B'is created stemming from peptides in B: ^^ᇱ1}where ANCHOR-MODIFIED(b,c) returns a set of all 204– 1 anchor-modified peptides stemming from b with all possible modifications to the amino acids at P1, P4, P6, and P9 of the 9-mer binding core c. Thus, for each base peptide b, the heteroclitic set B' contains all anchor-modified peptides b' with modifications to all unique cores of b identified for any HLA alleles that potentially present b with a valid core position as indicated by scoring matrix S2.

[0563] Next, all heteroclitic candidate peptides (e.g., modified peptides) in B' are scored against all HLA alleles in ℋ for predicted display using SCOREDISPLAY (with threshold τ2= 50 nM), and store results in binary |B'| × |ℋ| matrix ^^^ᇱ: ^^^ᇱ[ ^^ᇱ, ℎ] = ScoreDisplay( ^^ᇱ,ℎ) ∀ ^^ᇱ∈ ^^ᇱ,ℎ ∈ ℋ - 104 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0564] For each (heteroclitic peptide, HLA allele) pair (b',h), identify / predict the 9-mer binding core using FINDCORE. The predicted binding core is recorded in a matrix C': ^^ᇱ[^^ᇱ,ℎ]= FINDCORE(^^ᇱ, ℎ)∀ ^^ᇱ∈ ^^ᇱ,ℎ ∈ ℋ

[0565] An updated scoring matrix ^^ଶᇱis computed for heteroclitic peptides conditioned on the identified binding cores of a heteroclitic and base peptides occurring at the same offset by a particular HLA: ^^ଶᇱℋwhere each heteroclitic peptide b' ∈ B' is a mutation of base peptide b ∈ B. This condition enforces the binding core of the heteroclitic peptide b' to be at the same relative position as the base peptide b, and, implicitly, enforces that the target residue Ptstill falls in a non-anchor position within the 9-mer binding core (Step 3).

[0566] An updated scoring matrix ^^ଷᇱis computed for heteroclitic peptides conditioned on the potential presentation of the corresponding base peptides by each HLA: ^^ଷᇱℋwhere each heteroclitic peptide b' ∈ B' is a mutation of base peptide b ∈ B. This condition enforces that if h was not predicted to display b, then all heteroclitic peptides b' derived from b will not be displayed by h (even if h would otherwise be predicted to display b').

[0567] OptiVax-Robust is used to design a final peptide set (e.g., third peptide set) from the union of base peptides and heteroclitic peptides B ∪ B' (with corresponding scoring matrices S2 and ^^ଷᇱfor B and B', respectively). OptiVax will output m sets ^^^for s ∈ [1, …, m] where m is the largest vaccine size requested from OptiVax. Let ^^^denote the compact set of vaccine peptides output by OptiVax containing k peptides. Note that ^^^ା^is not necessarily a superset of ^^^. (In alternate embodiments, OptiVax can be used to augment the base set B with peptides from B' using scoring matrix ^^ଶᇱto have OptiVax return setand the final vaccine set ^^^ା|^|consists of peptides B ∪ ^^^.)

[0568] In some embodiments, this procedure is repeated independently for each single target of interest, and the resulting independent vaccine sets can be merged into a combined vaccine as described below. - 105 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 MHC Class I or Class II Vaccine Design Method Prioritizing Peptide Conservation

[0569] In some embodiments, peptide sequences that are more conserved across strains, species, or other protein sources of interest are prioritized for vaccine inclusion. In some embodiments, a set of related protein sequences called protein variants are considered for vaccine design. A protein variant is one instance of a family of protein sequences, and protein variants can be sequences from various species, pathogen strains (e.g., viral strains), or other variations considered for vaccine design. In some embodiments, each considered protein variant has an associated probability called a protein variant probability, where the sum of all protein variant probabilities for the supplied set of protein variants is one. In some embodiments, multiple proteins of interest can be considered for the design of a single vaccine using an MHC Class I or Class II vaccine design method prioritizing peptide conservation. In these embodiments, protein variants for all proteins of interest are collectively considered for generating candidate peptides. In some embodiments, the protein variant probabilities across all of the considered multiple proteins sum to one. The methods disclosed herein can be used to design an immunogenic composition, such as a vaccine, based on any protein variant, including variants that have yet to be discovered or future emerging variants (e.g., new virus species or strains).

[0570] A set of candidate peptides are created from each protein variant using a sliding window method that parses the protein variant into peptide sequences. In some embodiments, for MHC Class I, 8-mers, 9-mers, 10-mers, and / or 11-mers are produced, but this process can be performed with any desired window lengths and the resulting peptide sets can be combined. In some embodiments, for MHC Class I, only 9-mers are produced. In some embodiments, for MHC Class II, all windowed peptides of length 13–25 are produced, but this process can be performed using any desired window lengths (e.g., only 15-mers). In some embodiments, peptides that are predicted to be glycosylated in a given protein variant are removed and not considered for that variant as described in Liu et al. (2020a), which is incorporated by reference herein in its entirety.

[0571] In some embodiments, for each generated peptide sequence (MHC Class I or Class II) conservation is defined as the fraction of input protein variants where the peptide sequence occurs. For example, if a given 9-mer peptide sequence occurs in the peptides generated from 90% of the protein variants provided as input, its conservation is .90. In some embodiments, conservation is defined for each generated peptide sequence (MHC Class I or Class II) as the - 106 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 sum of the protein variant frequencies where the peptide sequence occurs. For example, if a given 9-mer peptide sequence occurs in the peptides generated from protein variants with protein variant probabilities of 0.10 and 0.20, its conservation is 0.30. In some embodiments, this functionality is implemented by a COMPUTECONSERVATION function that computes the sum of the frequencies of the protein variants that contain a peptide sequence. In some embodiments, when protein variants are not sufficient for computing expected future conservation, a method of predicting conservation can be used to implement COMPUTECONSERVATION, such as the one disclosed in Hie et al. (2021), which is incorporated by reference herein in its entirety.

[0572] In some embodiments, vaccine design considers conservation by prioritizing peptides for vaccine inclusion that are more conserved than others to meet a desired vaccine performance metric. In some embodiments, the vaccine design method attempts to first design a vaccine with candidate peptides that all meet a first conservation threshold, and if the desired vaccine performance is not met, it iteratively adds additional peptides with less stringent conservation to attempt to meet the desired vaccine performance metric. In some embodiments, vaccine design prioritizing conservation proceeds by setting a vaccine design D to be an empty set, and then performing the steps of: (1) selecting candidate peptides in which each peptide passes a conservation threshold to create a candidate peptide set and is not in D, (2) selecting vaccine designs having varying peptide numbers / combinations from this candidate set to optimize a vaccine performance metric using methods disclosed herein for MHC Class I or Class II vaccine design to augment the vaccine design contained in D (one implementation of vaccine augmentation is described in (Liu et al., 2021), incorporated by reference in its entirety herein), (3) selecting the smallest vaccine peptide set design from Step 2 that either meets the desired vaccine performance metric or where adding one more peptide to the selected set does not provide a desired minimum improvement in the vaccine performance metric, (4) if a vaccine peptide set was found in Step 3, adding the vaccine peptide set design from Step 3 to the vaccine design D, and (5) determining whether the vaccine design D meets a desired vaccine performance metric objective, and if so, return vaccine design D as the final vaccine design. If at Step 5, the vaccine design D fails to meet the desired vaccine performance metric objective, the computation continues with the following steps: (6) setting an updated conservation threshold to be lower than the current conservation threshold (less constrained) and (7) repeating the process starting from Step 1, retaining the current vaccine design D and current candidate set, until either a desired vaccine performance metric objective is reached at Step 5, or the updated conservation threshold is lower than a minimum desired conservation threshold at Step - 107 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 6. If on any iteration the updated conservation threshold is lower than a minimum desired conservation threshold, the latest version of vaccine design D will be used as the final vaccine design. When the process completes, the final vaccine design D includes all of the peptides that can be used in a vaccine.

[0573] In some embodiments, MHC class I or class II vaccine design procedures consist of the following computational steps.

[0574] In some embodiments, the inputs for the computation are: sequence of protein variant j of length nj. ^^^,^denotes the amino acid at position i of protein variant j, where ^^ ∈ [1, … ^^] and a is the number of protein variants ^^^: Protein variant probability of protein variant ^^^tj: Position in protein variant ^^^of the target mutation ^^ ∈[1, … ^^]D: The vaccine design, initialized to the empty set ∅ s: Substitution mutation ^^ ∈[^^ ^^ ^^ ^^, ^^ ^^ ^^ ^^ ^^]is true if the mutation is a substitution, and false if the mutation is a deletion or insertion or the peptide does not contain a mutation. When the mutation is a deletion or insertion then t indicates the position immediately before the deletion or insertion. ^^^: Initial conservation level of peptides ^^^: Current conservation threshold ^^ଶ: Change in conservation level on each iteration ^^^: Minimum final conservationv: Target vaccine performance metric^^ௗ: Minimum change in vaccine performance metric to increase vaccine size N: Parameter for EvalVax and OptiVax objective function. Specifies minimum number of predicted per-individual hits for population coverage objective to consider the individual covered. Default = 1 (computes P(n ≥ 1) population coverage). COMPUTECONSERVATION : ^^ × ^^ × ^^ → ℝ: In some embodiments, computes the fraction of sets Xjthat contain sequence S. In some embodiments, sums all the Ojwhere sequence S appears in Xj- 108 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0575] The protein variant sequences ^^^are used to produce windowed peptides that span the protein sequence(s) starting at each location m with a peptide length of k residues. The result is the set Xjthat contains all of the peptide sequences in protein variant Pj. ^^^,^…^ା(^ି^)only produces a sequence when the subscripts are within the range of the defined protein Pj. In some embodiments for MHC Class I, k is chosen to produce 8-mers, 9-mers, 10-mers, and / or 11-mers, but this process can be performed with any desired window lengths and the resulting peptide sets can be combined. In some embodiments for MHC Class I, only 9-mers are produced. In some embodiments for MHC Class II, all windowed peptides of length 13–25 are extracted, but this process can be performed using any desired window lengths (e.g., only 15-mers). , ^^^ ^

[0576] In some embodiments for MHC Class I, the second condition m ≠ {t – (k-1), t – 1} excludes peptides where the mutation at t is in positions P2 or Pk of the windowed k-mer peptide (i.e., the anchor positions) and the mutation is a substitution for MHC Class I design. MHC Class II anchor positions are filtered in the MHC Class II design method.

[0577] In some embodiments, the methods disclosed herein are used to create the set of all peptides B that occur in any input protein variant. ^^ = ^ ^^^^∈[^,…^]^^ = | ^^|

[0578] In some embodiments, for each peptide ^^௪in ^^ its conservation metric Cw is computed using COMPUTECONSERVATION Cw= COMPUTECONSERVATION[ ^^௪, X, O]

[0579] In some embodiments, the current conservation threshold is then set to the initial conservation threshold: ^^^= ^^^- 109 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024

[0580] In some embodiments, at Step 1, candidate peptides are selected where each peptide passes a conservation threshold to create a candidate peptide set and is not already in D. A set of peptide candidates ^^ is defined such that each candidate peptide meets the current conservation threshold c^and the peptide candidate is not already in D. D is set to be empty (0 peptides) on the first iteration of the computational steps. ^^ =⋃ ௪∈[^,…,௭]^^௪^^ℎ ^^ ^^ ^^ ^^௪≥ ^^^^^ ^^ ^^ ^^௪∉ ^^

[0581] In some embodiments, at Step 2, vaccine designs are selected having varying peptide numbers / combinations from the candidate set to optimize a vaccine performance metric using methods disclosed herein for MHC Class I or Class II vaccine design to augment the vaccine design contained in D. The peptide set ^^ is provided to “MHC Class I Vaccine Design Procedure with Defined Peptide Set ^^” for MHC Class I and “MHC Class II Vaccine Design Procedure with Defined Peptide Set ^^” for MHC Class II. The peptide set ^^ is provided as the set of candidates to augment the set D. Both the set ^^ and D are provided to OptiVax which uses D as the fixed starting set and augments D with peptides from the set ^^ using vaccine augmentation as described in Liu et al., 2021, incorporated by reference in its entirety herein. OptiVax-Robust is used to augment the set D with peptides from ^^ using the scoring matrices as defined in “MHC Class I Vaccine Design Procedure with Defined Peptide Set ^^” for MHC Class I and “MHC Class II Vaccine Design Procedure with Defined Peptide Set ^^” for MHC Class II, and returns sets ^^^where each set ^^^is a compact set of vaccine peptides output by OptiVax containing s peptides. In some embodiments, the steps to modify anchor positions are not utilized in the MHC Class I or MHC Class II vaccine design methods and only the base peptides B are utilized for vaccine design. In some embodiments, positions in addition to anchor positions are modified in the MHC Class I or MHC Class II vaccine design methods utilized to create B’.

[0582] In some embodiments, at Step 3, the smallest vaccine peptide set design is selected from Step 2 that either meets the desired vaccine performance metric or where adding one more peptide to the selected set does not provide a desired minimum improvement in the vaccine performance metric. A vaccine design ^^^is chosen that meets minimum requirements. In some embodiments, the vaccine design ^^^is chosen with the value s chosen to be the minimum value of s such that the difference in vaccine performance between D ∪ ^^^and D ∪ ^^^ା^is less than v^. In some embodiments, the value s is chosen to be the minimum value such that the - 110 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 vaccine performance metric of D ∪ meets the final vaccine performance metric v. In some embodiments, ^^^ା^is not necessarily a superset of ^^^.

[0583] In some embodiments, at Step 4, if a vaccine peptide set was found in Step 3, it is added to the vaccine peptide set design D. If an acceptable vaccine design ^^^was found in Step 4, the vaccine design set D is updated to consist of D ∪ ^^^.

[0584] In some embodiments, at Step 5, it is determined whether the vaccine design D meets a desired vaccine performance metric objective. If the vaccine design set D meets the final vaccine performance design metric v, D is returned as the final design.

[0585] In some embodiments, at Step 6, the conservation threshold is updated to be lower than the current conservation threshold (less constrained). If the vaccine design set D does not meet the final vaccine performance design metric v, ^^^is reduced: ^^^= ^^^− ^^ଶ

[0586] In some embodiments, at Step 7, the process is repeated starting at Step 1, retaining the current vaccine design D and current candidate set, until either a desired vaccine performance metric objective is reached at Step 5, or the updated conservation threshold at Step 6 is lower than a minimum desired conservation threshold. If ^^^< ^^^then design set D is returned as the final vaccine design. If not, the method returns to Step 1 and repeats all subsequent steps.

[0587] In some embodiments, this procedure is repeated independently for each pathogen gene variant or target variant of interest, and the resulting independent vaccine sets can be merged into a combined vaccine. In some embodiments, this procedure is repeated independently for each single protein target of interest (e.g., influenza, cancer neoantigen, etc.), and the resulting independent vaccine sets can be merged into a combined vaccine. Methods for combining multiple vaccines

[0588] The above-described methods will produce an optimized target peptide set (e.g., third peptide set) for one or more individual targets. In some embodiments, a method is provided for designing separate vaccines for MHC class I and class II-based immunity for multiple targets (e.g., two or more targets such as KRAS G12D and KRAS G12V).

[0589] In some embodiments, a method is disclosed for producing a combined peptide vaccine for multiple targets by using a table of presentations for a disease that is based on - 111 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 empirical data from sources such as the Cancer Genome Atlas (TCGA). In some embodiments, a method is disclosed for producing a combined peptide vaccine for multiple strains of a pathogen (e.g., influenza) that is based on empirical data of strain prevalence where prevalence is used to represent the probability of the presentation of a given strain. In some embodiments, for a given strain presentation, the probabilities of each pathogen protein target are identical and sum to one. In some embodiments, for a given strain presentation, the probabilities of each pathogen protein target vary based on the observed or predicted immunogenicity of each protein (e.g., based on the expression of the pathogen protein or its processing).

[0590] FIG.6 shows various embodiment for factoring in disease presentation type (e.g., pancreatic cancer, colorectal cancer, and skin cancer) by probability, for each disease presentation, of target presented for various mutation targets (e.g., KRAS G12D, KRAS G12V, and KRAS G12R). A presentation is a unique set of targets that are presented by one form of a disease (e.g., distinct type of cancer or cancer indication as shown in FIG.6). For each presentation, FIG.6 shows an example of the probability of that presentation, and the probability that a given target is observed. For a given presentation, there can be one or more targets, each having a probability. In some embodiments, the method for multi-target vaccine design will allocate peptide resources for inducing disease immunity based on the presentation and respective target probabilities as shown in FIG.6, for example. In some embodiments, presentations correspond to the prevalence of targets in different human populations or different risk groups. The probability of a target in a population is computed by summing for each possible presentation the probability of that presentation times the probability of the target in that presentation. FIG.6 shows weights used for merging individual vaccines for each target (row) into combined vaccines for each disease indication (column). Values indicate the observed fraction of cases containing each target mutation. Data are from The Cancer Genome Atlas (TCGA). For each disease indication, TCGA data are filtered to cases where the Primary Site is the indication.

[0591] In some embodiments, presentations are used to represent different strains of pathogens, where the probability of a presentation is based on the observed prevalence of a given pathogen strain. In some embodiments, for a given strain presentation, the probabilities of each pathogen protein target are identical and sum to one. In some embodiments, for a given strain presentation, the probabilities of each pathogen protein target vary based upon the observed or predicted immunogenicity of each pathogen protein (e.g., based on the expression of - 112 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 the pathogen protein or its processing). In some embodiments, presentation probabilities are used to create a combined vaccine for more than one pathogen strain, where vaccines for each individual strain are combined using the prevalence probabilities of each strain. In some embodiments, the immunogenic importance of each pathogen protein is considered by assigning different presentation probabilities to different pathogen proteins that are used as vaccine targets, and the resulting target specific vaccines are combined using these probabilities.

[0592] In some embodiments, the same vaccine design will be generated for mutations to different proteins when the base peptides generated by the mutations to the different proteins are identical. For example, in some embodiments of base peptide selection the following mutations have identical vaccine designs because they share the same set of base peptides: HRAS Q61K, NRAS Q61K, and KRAS Q61K; HRAS Q61L, NRAS Q61L, and KRAS Q61L; HRAS Q61R, NRAS Q61R, and KRAS Q61R. Referring to FIG.6, in some embodiments when two mutations have identical individual vaccine designs their presentation specific probabilities are added when weighting the individual vaccine design for inclusion in a combined vaccine as described below (e.g., for Thyroid Cancer NRAS Q61R and HRAS Q61R).

[0593] Referring to FIG.7, in some embodiments, the method first includes designing an individual peptide vaccine for each target to create a combined vaccine design for multiple targets. This initially results in sets of target-specific vaccine designs. In some embodiments, the marginal predicted vaccine performance of each target-specific vaccine at size k is defined by predicted vaccine performance at size k minus the predicted vaccine performance of the vaccine at size k minus one (see FIGs.3-4). The composition of a vaccine may change as the number of peptides used in the vaccine increases, and thus for computing contributions to a combined vaccine the marginal predicted vaccine performance of each target-specific vaccine is used instead of a specific set of peptides.

[0594] In some embodiments, the weighted marginal predicted vaccine performance of a target-specific vaccine design for each target specific vaccine size is computed as shown in FIG. 8. For a given target specific vaccine size, its weighted predicted vaccine performance is computed by multiplying its predicted vaccine performance times the probability of the target in the population (e.g., by using values as shown in FIG.6). The marginal weighted predicted vaccine performance for a target specific vaccine is its weighted coverage at size k minus its coverage a size k minus one (e.g., see FIGs.3-4). The marginal weighted predicted vaccine performance of a target specific vaccine of size one is its weighted predicted vaccine - 113 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 performance. The marginal weighted predicted vaccine performances for all vaccines are combined into a single list, and the combined list is sorted from largest to least by the weighted marginal predicted vaccine performances of the target specific vaccines as shown in FIG.7. The combined vaccine of size n is then determined by the first n elements of this list. The peptides for the combined vaccine are determined by the individual peptide target vaccines whose sizes add to n and whose weighted predicted vaccine performances sums to the same sum as the first n elements of the sorted list. This maximizes the predicted vaccine performance of the combined vaccine of size n.

[0595] In some embodiments, the combined multiple target vaccine can be designed on its overall predicted coverage for the disease described depending on the presentation table used (e.g., see FIG.6), by its predicted coverage for a specific indication, and / or by its predicted coverage for a specific target by adjusting the weighting used for predicted vaccine performance accordingly. Once a desired level of coverage is selected, the peptides of the combined vaccine are determined by the contributions of target-specific designs. For example, if the combined vaccine includes a target-specific vaccine of size k, then the vaccine peptides for this target at size k are used in the combined vaccine.

[0596] Table 1 (below) contains the peptides present in independent (single target) MHC class I vaccine designs. Table 2 (below) contains the contains the peptides present in independent (single target) MHC class II vaccine designs. For alternate embodiments, the Sequence Listing provides heteroclitic peptides useful in MHC class I vaccines and MHC class II vaccines for the mutated proteins, specific protein mutations, and indications in Tables 1 and 2. Any subset of the individual / single target vaccines for MHC class I and class II can be combined to create a vaccine for two or more multiple targets. Combined Vaccine Design Procedure

[0597] In some embodiments, the procedure described herein is used to combine individual compact vaccines optimized for different targets into a single optimized combined vaccine.

[0598] In some embodiments, the computational inputs for the procedure are: ^^: Set of neoantigen or pathogenic targets of interest (e.g., KRAS G12D, KRAS G12V, KRAS G12R) - 114 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 ^^: Vaccine sets optimized individually for each target. Let ^^௧,^denote the optimal vaccine set of exactly k peptides for target t ∈ ^^ (e.g., as computed by the procedures describe above). Note that ^^௧,^ା^may not necessarily be a superset of ^^௧,^. W: ^^ → [0,1] : Target weighting function mapping each target t ∈ ^^ to a probability or weight of t in a particular presentation of interest (e.g., pancreatic cancer; see FIG.6, for example). POPULATIONCOVERAGE: ^^ → [0,1]: Function mapping a peptide set into population coverage (e.g., EvalVax). This function may also take as input additional parameters, including HLA haplotype frequencies and a minimum per-individual number of peptide- HLA hits N (here, we compute coverage as P(n ≥ 1) using EvalVax-Robust).

[0599] At Step 1, for each target t (individually) compute optimized vaccines of sizes 1 to m (1 to m peptides; m is the largest vaccine size used for the computation) as the sets ^^௧,^where k denotes the size of the vaccine. Then, compute the vaccine performance for each vaccine size. For each target t (individually) and vaccine size (peptide count) k, the unweighted population coverage ^^௧,^is computed: ^^௧,^= ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^൫ ^^௧,^൯ ∀ ^^ ∈ ^^, ^^ In some embodiments, for each target t, ct,kis gen...

Claims

Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 What is claimed is:

1. A composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 17 to 176, and SEQ ID NOs: 178 to 184.

2. The composition of claim 1, wherein the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides.

3. The composition of claim 2, wherein an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject.

4. The composition of claim 3, wherein the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele.

5. The composition of claim 1, wherein each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a CTNNB1 protein.

6. The composition of claim 5, wherein the fragment of the CTNNB1 protein comprises a mutation selected from the group consisting of CTNNB1 D32G, CTNNB1 G34E, CTNNB1 S33C, CTNNB1 S33F, CTNNB1 S37C, CTNNB1 S37F, CTNNB1 S45F, CTNNB1 S45P, CTNNB1 T41A, and CTNNB1 T41I.

7. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 1, wherein the composition is an immunogenic composition. - 594 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 8. The composition of claim 1, wherein the at least two amino acid sequences are SEQ ID NOs: 103 and 104.

9. A composition comprising one or more polynucleotides encoding at least two amino acid sequences selected from the group consisting of SEQ ID NOs: 185 to 207 and SEQ ID NOs: 209 to 222.

10. The composition of claim 9, wherein the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides.

11. The composition of claim 10, wherein an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject.

12. The composition of claim 11, wherein the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele.

13. The composition of claim 9, wherein each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a KRAS protein.

14. The composition of claim 13, wherein the fragment of the KRAS protein comprises a mutation selected from the group consisting of KRAS A146T, KRAS A146V, and KRAS Q61H.

15. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 9, wherein the composition is an immunogenic composition. - 595 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 16. The composition of claim 9, wherein the at least two amino acid sequences are SEQ ID NOs: 203 and 204.

17. A composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 223 to 240, SEQ ID NOs: 243 to 293, SEQ ID NOs: 295 to 315, SEQ ID NOs: 317 to 329, SEQ ID NOs: 331 to 339, and SEQ ID NOs: 341 to 386.

18. The composition of claim 17, wherein the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides.

19. The composition of claim 18, wherein an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject.

20. The composition of claim 19, wherein the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele.

21. The composition of claim 17, wherein each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a PIK3CA protein.

22. The composition of claim 21, wherein the fragment of the PIK3CA protein comprises a mutation selected from the group consisting of PIK3CA C420R, PIK3CA E453K, PIK3CA E545A, PIK3CA E726K, PIK3CA G118D, PIK3CA H1047L, PIK3CA N345K, PIK3CA Q546K, PIK3CA Q546R, and PIK3CA R108H. - 596 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 23. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 17, wherein the composition is an immunogenic composition.

24. The composition of claim 17, wherein the at least two amino acid sequences are SEQ ID NOs: 308 and 309.

25. A composition comprising one or more polynucleotides encoding at least two amino acid sequences, wherein the at least two amino acid sequences are selected from the group consisting of SEQ ID NOs: 387 to 445, SEQ ID NOs: 447 to 472, SEQ ID NOs: 474 to 480, SEQ ID NOs: 482 to 500, SEQ ID NOs: 502 to 544, SEQ ID NOs: 546 to 550, SEQ ID NOs: 552 to 564, SEQ ID NOs: 566 to 582, SEQ ID NO: 584, SEQ ID NOs: 586 to 605, SEQ ID NOs: 607 to 633, SEQ ID NOs: 638 to 656, SEQ ID NOs: 659 to 664, and SEQ ID NOs: 666 to 794.

26. The composition of claim 25, wherein the one or more polynucleotides are contained in a construct for in vivo expression in a subject of at least two peptides encoded by the one or more polynucleotides.

27. The composition of claim 26, wherein an administration of the one or more polynucleotides causes the at least two peptides encoded by the one or more polynucleotides to be displayed by a HLA class I molecule in the subject.

28. The composition of claim 27, wherein the administration of the one or more polynucleotides causes: a first peptide of the at least two peptides to be displayed by a first plurality of HLA class I alleles; and a second peptide of the at least two peptides to be displayed by a second plurality of HLA class I alleles, wherein the first plurality of HLA class I alleles and the second plurality of HLA class I alleles differ by at least one HLA class I allele.

29. The composition of claim 25, wherein each of the at least two amino acid sequences comprises a heteroclitic modification of a fragment of a TP53 protein. - 597 - ActiveUS 205953775Attorney Docket No. 2215269.00133WO1 Date of Electronic Deposit: September 16, 2024 30. The composition of claim 29, wherein the fragment of the TP53 protein comprises a mutation selected from the group consisting of TP53 C176F, TP53 C176Y, TP53 C238Y, TP53 C275Y, TP53 E285K, TP53 G245S, TP53 G245V, TP53 H179Y, TP53 H193R, TP53 I195T, TP53 K132E, TP53 K132N, TP53 P151S, TP53 R213L, TP53 R249M, TP53 R249S, TP53 R273L, TP53 R280K, TP53 S127Y, TP53 S241F, TP53 V157F, TP53 V272M, TP53 Y163C, TP53 Y205C, and TP53 Y234C.

31. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 25, wherein the composition is an immunogenic composition.

32. The composition of claim 25, wherein the at least two amino acid sequences are SEQ ID NOs: 474 and 475. - 598 - ActiveUS 205953775

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