Neoantigens and their uses

Abstract

To provide a neoantigen, and to provide uses thereof.SOLUTION: The disclosure herein relates to immunotherapeutic compositions comprising immunotherapeutic peptides comprising neoepitopes. Also disclosed herein are polynucleotides encoding the immunotherapeutic peptides. Also disclosed herein are methods of synthesis of immunotherapeutic peptides comprising neoepitopes and use of the immunotherapeutic compositions including methods of treatment. In one aspect, provided herein is a method of treating a subject having cancer, comprising administering to the subject a pharmaceutical composition of any one of the above aspects.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 687,191, filed June 19, 2018, U.S. Provisional Patent Application No. 62 / 702,567, filed July 24, 2018, U.S. Provisional Patent Application No. 62 / 726,804, filed September 4, 2018, U.S. Provisional Patent Application No. 62 / 789,162, filed January 7, 2019, U.S. Provisional Patent Application No. 62 / 801,981, filed February 6, 2019, U.S. Provisional Patent Application No. 62 / 800,700, filed February 4, 2019, and U.S. Provisional Patent Application No. 62 / 800,792, filed February 4, 2019, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapy exploits the fact that cancer cells often have molecules on their surface that can be detected by the immune system; these molecules, known as tumor antigens, are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing antitumor responses and involves the use of monoclonal antibodies, lymphocytes, and cytokines. Tumor vaccines generally consist of tumor antigens and immune stimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. One of the serious obstacles to the development of curative and tumor-specific immunotherapy is the identification and selection of highly specific, defined tumor antigens to avoid autoimmunity.

[0003] Tumor neoantigens, which arise as a result of genetic alterations in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific class of antigens and can be patient-specific or shared. Tumor neoantigens are unique to tumor cells because the mutations and their corresponding proteins are present only in that tumor. They also evade central tolerance and are therefore more likely to be immunogenic. Therefore, tumor neoantigens provide excellent targets for immune recognition, including both humoral and cellular immunity. However, tumor neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to the technical challenges of identifying them, optimizing antigen selection, and producing neoantigens for use in vaccines or immunogenic compositions. Therefore, there is still a need for the development of additional cancer therapeutics. Incorporation by Reference

[0004] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, provided herein is a pharmaceutical composition comprising: (a) at least one polypeptide comprising a first mutant GATA3 peptide sequence and a second mutant GATA3 peptide sequence, or a pharmaceutically acceptable salt thereof, wherein (i) each of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprises at least 8 consecutive amino acids of SEQ ID NO: 1; and (ii) the C-terminal sequence of the first mutant GATA3 peptide sequence overlaps with the N-terminal sequence of the second mutant GATA3 peptide sequence, and the at least 8 consecutive amino acids of SEQ ID NO: 1 comprise at least one amino acid of the sequence: PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); or (b) at least one polynucleotide comprising a sequence encoding the at least one polypeptide, or a pharmaceutically acceptable salt thereof.

[0006] In some embodiments, the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprise at least 8 contiguous amino acids of SEQ ID NO: 2. In some embodiments, the first mutant GATA3 peptide sequence and the second mutant peptide sequence comprise at least 8 contiguous amino acids of SEQ ID NO: 2.

[0007] In some embodiments, the at least 8 contiguous amino acids of SEQ ID NO:2 comprise at least 8 contiguous amino acids of the sequence: PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGL (SEQ ID NO:3).

[0008] In some embodiments, the at least 8 contiguous amino acids of SEQ ID NO:2 include at least one amino acid of the sequence: EPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO:4).

[0009] In some embodiments, at least one of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprises at least 14 mutant amino acids. In some embodiments, the at least one polypeptide comprises at least three mutant GATA3 peptide sequences. In some embodiments, the at least one polypeptide comprises at least two polypeptides. In some embodiments, the at least one polypeptide further comprises a third mutant GATA3 peptide sequence, wherein the third mutant GATA3 peptide sequence comprises at least 8 consecutive amino acids of SEQ ID NO: 1, and the at least 8 consecutive amino acids of SEQ ID NO: 1 comprise at least one amino acid of the sequence of SEQ ID NO: 2. In some embodiments, the third mutant GATA3 peptide comprises at least 8 consecutive amino acids of SEQ ID NO: 2.

[0010] In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele. In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by (a) an HLA-A02:01 allele and an HLA-A24:02 allele, (b) an HLA-A02:01 allele and an HLA-B08:01 allele, (c) an HLA-A24:02 allele and an HLA-B08:01 allele, or (d) an HLA-A02:01 allele, an HLA-A24:02 allele, and an HLA-B08:01 allele. In some embodiments, (a) the first mutant GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele; (b) the second GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele; and the first mutant GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA allele different from the second mutant GATA3 peptide sequence.

[0011] In some embodiments, at least one of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence binds to a protein encoded by an HLA allele with an affinity of less than 500 nM.

[0012] In some embodiments, at least one of the first mutant GATA3 peptide sequence and the second mutant peptide sequence stably binds to a protein encoded by an HLA allele for more than 1 hour.

[0013] In some embodiments, at least one polypeptide comprises at least one of the following sequences: (a) TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI, (b) MFLKAESKI and / or YMFLKAESKI, (c) VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR, (d) FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL, and / or (e) IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI.

[0014] In some embodiments, at least one polypeptide comprises at least two of the following sequences: (a) TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI, (b) MFLKAESKI and / or YMFLKAESKI, (c) VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR, (d) FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL, and / or (e) IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI.

[0015] In some embodiments, the mutant GATA3 peptide sequence is selected from the group consisting of: (a) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (b); (b) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (c); (c) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (d); (d) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (e); (e) the first mutant GATA3 peptide sequence from (b) and the second mutant GATA3 peptide sequence from (c). (f) a first mutant GATA3 peptide sequence from (b) and a second mutant GATA3 peptide sequence from (d); (g) a first mutant GATA3 peptide sequence from (b) and a second mutant GATA3 peptide sequence from (e); (h) a first mutant GATA3 peptide sequence from (c) and a second mutant GATA3 peptide sequence from (d); (i) a first mutant GATA3 peptide sequence from (c) and a second mutant GATA3 peptide sequence from (e); or (j) a first mutant GATA3 peptide sequence from (d) and a second mutant GATA3 peptide sequence from (e).

[0016] In some embodiments, the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprise a peptide in Table 5 and / or Table 6. In some embodiments, the first mutant GATA3 peptide sequence comprises a first neoepitope of a GATA3 protein and the second peptide mutant GATA3 peptide sequence comprises a second neoepitope of a mutant GATA protein, the first mutant GATA3 peptide sequence is different from the second mutant GATA3 peptide sequence, the first neoepitope comprises at least one mutant amino acid, and the second neoepitope comprises the same mutant amino acid.

[0017] In some embodiments, each of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprising at least 8 consecutive amino acids comprises the amino acid sequence [Xaa] F -[Xaa] N -[Xaa] C or [Xaa] N -[Xaa] C -[Xaa] F wherein each Xaa is an amino acid, [Xaa] N and [Xaa] C Each contains an amino acid sequence encoded by a different portion of the GATA3 gene, [Xaa] F is any amino acid sequence, [Xaa] N is encoded in a non-wild-type reading frame of the GATA3 gene and is [Xaa] C contains at least one mutant amino acid and is encoded in a non-wild-type reading frame of the GATA3 gene, N is an integer of 0 to 100, C is an integer of 1 to 100, F is an integer of 0 to 100, and the sum of N and M is at least 8.

[0018] In some embodiments, [Xaa] F each Xaa is a lysine residue, and F is an integer from 1 to 100, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, F is 3, 4, or 5.

[0019] In some embodiments, each of the mutant GATA3 peptide sequences is present at a concentration of at least 50 μg / mL to 400 μg / mL. In some embodiments, the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprise a sequence in Table 1 or Table 2. In some embodiments, the composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the adjuvant is poly-ICLC.

[0020] In one aspect, a pharmaceutical composition is provided comprising one or more mutant GATA3 peptide sequences, wherein the one or more mutant GATA3 peptide sequences comprise a sequence selected from the group consisting of ESKIMFATLQRSSL, KPKRDGYMFLKAESKI, SMLTGPPARVPAVPFDLH, EPCSMLTGPPARVPAVPFDLH, LHFCRSSIMKPKRDGYMFLKAESKI, GPPARVPAVPFDLHFCRSSIMKPKRD, and KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH.

[0021] In some embodiments, the one or more mutant GATA3 peptide sequences are ESKIMFATLQRSSL. In some embodiments, the one or more mutant GATA3 peptide sequences are KPKRDGYMFLKAESKI. In some embodiments, the one or more mutant GATA3 peptide sequences are SMLTGPPARVPAVPFDLH. In some embodiments, the one or more mutant GATA3 peptide sequences are EPCSMLTGPPARVPAVPFDLH. In some embodiments, the one or more mutant GATA3 peptide sequences are LHFCRSSIMKPKRDGYMFLKAESKI. In some embodiments, the one or more mutant GATA3 peptide sequences are GPPARVPAVPFDLHFCRSSIMKPKRD. In some embodiments, the one or more mutant GATA3 peptide sequences are KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH.

[0022] In some embodiments, the pharmaceutical composition comprises a pH adjusting agent present at a concentration of 0.1 mM to 1 mM, hi some embodiments, the pharmaceutical composition comprises a pH adjusting agent present at a concentration of 1 mM to 10 mM.

[0023] In one aspect, the present disclosure provides a method for synthesizing a GATA3 peptide, wherein the peptide comprises a sequence of at least two consecutive amino acids selected from the group consisting of Xaa-Cys, Xaa-Ser, and Xaa-Thr (wherein Xaa is any amino acid), and the method comprises: (a) coupling at least one dipeptide or a derivative thereof with an amino acid or a derivative thereof of the GATA3 peptide or its derivative to obtain a pseudoproline-containing GATA3 peptide or its derivative, wherein the dipeptide or its derivative comprises a pseudoproline moiety; (b) coupling one or more selected amino acids, small peptides, or derivatives thereof with the pseudoproline-containing GATA3 peptide or its derivative; and (c) cleaving the pseudoproline-containing GATA3 peptide or its derivative from the resin. In some embodiments, the method comprises deprotecting the pseudoproline-containing GATA3 peptide or its derivative.

[0024] In some embodiments, the amino acid or derivative thereof to which at least one dipeptide or derivative thereof is coupled is selected from the group consisting of Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, Tyr, His, and Val. In some embodiments, the one or more selected amino acids, small peptides or derivatives thereof optionally coupled to the pseudoproline containing GATA3 peptide or derivative thereof are selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc- Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(t Bu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH.

[0025] In some embodiments, the N-terminal amino acid or derivative thereof of the GATA3 peptide or derivative thereof is selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc c-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH.

[0026] In some embodiments, the pseudoproline moiety is (a) Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH, (b) Fmoc-Ala-Thr(psi(Me,Me)pro)-OH, (c) Fmoc-Glu(OtBu)-Ser(psi(Me,Me)pro)-OH, (d) Fmoc-Leu-Thr(psi(Me,Me)pro)-OH, or (e) Fmoc-Leu-Cys(psi(Dmp,H)pro)-OH. In some embodiments, (a) Xaa-Ser is Ser-Ser, (b) Xaa-Ser is Glu-Ser, (c) Xaa-Thr is Ala-Thr, (d) Xaa-Thr is Leu-Thr, or (e) Xaa-Cys is Leu-Cys.

[0027] In one aspect, provided herein is a method of treating a subject having cancer, the method comprising administering to the subject a pharmaceutical composition of any one of the above aspects.

[0028] In one aspect, a method for identifying a subject having cancer as a candidate for a therapeutic agent comprises identifying the subject as expressing a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele; and wherein the therapeutic agent is (a) at least one polypeptide comprising one or more mutant GATA3 peptide sequences, each of the one or more mutant GATA3 peptide sequences comprising at least one mutant amino acid, and wherein the polypeptide is a GATA3 gene candidate of the cancer cell. Provided herein are methods comprising (a) at least one polypeptide, the fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in (b) at least one polypeptide, the fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in (c) at least one polynucleotide comprising a sequence encoding at least one polypeptide, wherein each of the one or more mutant GATA3 peptide sequences or a portion thereof binds to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele. In some embodiments, the method further comprises administering a therapeutic agent to the subject.

[0029] In one aspect, a method of treating a subject having cancer comprises administering to the subject a pharmaceutical composition; the pharmaceutical composition comprising: (a) at least one polypeptide comprising a first mutant GATA3 peptide sequence and a second mutant GATA3 peptide sequence, (i) the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence each comprising at least 8 consecutive amino acids of SEQ ID NO: 1; (ii) a C-terminal sequence of the first mutant GATA3 peptide sequence overlaps with an N-terminal sequence of the second mutant GATA3 peptide sequence; or (b) at least one polypeptide, wherein at least eight contiguous amino acids of at least one polypeptide comprise at least one amino acid of the sequence PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); or (b) at least one polynucleotide comprising a sequence encoding the at least one polypeptide; wherein the HLA alleles expressed by the subject are unknown at the time of administration.

[0030] In some embodiments, the at least 8 contiguous amino acids of SEQ ID NO: 1 include at least one amino acid of the sequence: PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2).

[0031] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL). In some embodiments, the subject has breast cancer that is resistant to anti-estrogen therapy, is MSI breast cancer, is metastatic breast cancer, is Her2-negative breast cancer, is Her2-positive breast cancer, is ER-negative breast cancer, is ER-positive breast cancer, is PR-positive breast cancer, is PR-negative breast cancer, or any combination thereof.

[0032] In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the method of the above aspects further comprises administering at least one additional therapeutic agent or therapy. In some embodiments, the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, an anti-CD40 agent, letrozole, fulvestrant, a PI3 kinase inhibitor, and / or a CDK4 / 6 inhibitor. In some embodiments, the at least one additional therapeutic agent is palbociclib, ribociclib, abemaciclib, seliciclib, dinaciclib, milciclib, roniciclib, atubeciclib, bliciclib, ribiciclib, seliciclib, trilaciclib, voruciclib, or any combination thereof.

[0033] In some embodiments, the at least one additional therapeutic agent is palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; alcyaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-aminothioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (lyubidin); flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atubeciclib; bliciclib; ribiciclib; trilaciclib (G1T28); or any combination thereof.

[0034] In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of wortmannin, demethoxyviridin, LY294002, hibiscone C, idelalisib, copanlisib, duvelisib, taselisib, perifosine, buparilisib, duvelisib, alpelisib (BYL719), umbralisib, (TGR1202), copanlisib (BAY80-6946), PX-866, dactolisib, CUDC-907, voxtalisib (S AR245409, XL765), CUDC-907, ME-401, IPI-549, SF1126, RP6530, INK1117, pictilisib (GDC-0941), XL147 (SAR245408), Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477 or AEZS-136.

[0035] In some embodiments, the cancer is recurrent or metastatic breast cancer. In some embodiments, the subject has experienced disease progression after endocrine therapy in combination with a CDK4 / 6 inhibitor, or the subject has not previously received systemic therapy. In some embodiments, the method includes determining the mutation status of the estrogen receptor gene in the subject's cells. In some embodiments, the cells are isolated cells or cells in which estrogen receptor expression is increased.

[0036] In some embodiments, provided herein are compositions comprising at least one polypeptide comprising one or more mutant GATA3 peptide sequences, each of which comprises at least one mutant amino acid and is a fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in the GATA3 gene of a cancer cell; at least one polynucleotide comprising a sequence encoding at least one polypeptide; one or more APCs comprising at least one polypeptide; or a T cell receptor (TCR) specific to a neoepitope of at least one polypeptide, in a complex with an HLA protein.

[0037] In some embodiments, the one or more mutant GATA3 peptide sequences comprise two or more mutant GATA3 peptide sequences, and each of the one or more mutant GATA3 peptide sequences comprises at least 8 consecutive amino acids of SEQ ID NO: 1 or 2.

[0038] In some embodiments, provided herein are compositions comprising at least one polypeptide comprising two or more mutant GATA3 peptide sequences, each of the two or more mutant GATA3 peptide sequences comprising at least 8 consecutive amino acids of SEQ ID NO: 1, and wherein the C-terminal sequence of the first GATA3 peptide sequence overlaps with the N-terminal sequence of the second GATA3 peptide sequence; at least one polynucleotide comprising a sequence encoding at least one polypeptide; one or more APCs comprising at least one polypeptide; or a T cell receptor (TCR) specific to a neoepitope of at least one polypeptide, forming a complex with an HLA protein.

[0039] In some embodiments, the mutant GATA3 peptide sequence comprises a fragment of a mutant GATA3 protein resulting from a frameshift mutation in the GATA3 gene of a cancer cell. In some embodiments, the at least 8 consecutive amino acids comprise at least one amino acid encoded by a GATA3 neo-ORF sequence. In some embodiments, the mutation in the GATA3 gene of a cancer cell is a frameshift mutation. In some embodiments, the mutation in the GATA3 gene of a cancer cell is a missense mutation, a splice site mutation, or a gene fusion mutation. In some embodiments, each of the mutant GATA3 peptide sequences comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mutant amino acids.

[0040] In some embodiments, at least one polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 mutant GATA3 peptide sequences. In some embodiments, at least one polypeptide comprises at least two polypeptides, or at least one polynucleotide comprises at least two polynucleotides. In some embodiments, at least one of the one or more GATA3 peptide sequences, or at least one of the two or more GATA3 peptide sequences, comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of a GATA3 protein. In some embodiments, at least two of the GATA3 peptide sequences comprise at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of a GATA3 protein.

[0041] In some embodiments, each of the GATA3 peptide sequences comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of a GATA3 protein. In some embodiments, at least one of the two or more mutant GATA3 peptide sequences comprises at least 8 consecutive amino acids of SEQ ID NO: 2. In some embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 of the two or more mutant GATA3 peptide sequences comprise at least 8 consecutive amino acids of SEQ ID NO: 2. In some embodiments, each of the two or more mutant GATA3 peptide sequences comprises at least 8 consecutive amino acids of SEQ ID NO: 2. In some embodiments, at least one of the two or more mutant GATA3 peptide sequences comprises at least 8 contiguous amino acids of SEQ ID NO:3.

[0042] In some embodiments, at least one of the at least 8 contiguous amino acids is an amino acid of SEQ ID NO: 4. In some embodiments, a contiguous amino acid of the at least 8 contiguous amino acids is not an amino acid of SEQ ID NO: 4. In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele. In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele and an HLA-A24:02 allele, an HLA-A02:01 allele and an HLA-B08:01 allele, an HLA-A24:02 allele and an HLA-B08:01 allele, or an HLA-A02:01 allele, an HLA-A24:02 allele and an HLA-B08:01 allele.

[0043] In some embodiments, the two or more mutant GATA3 peptide sequences include a first mutant GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele, and a second GATA3 peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele, wherein the first mutant GATA3 peptide sequence binds or is predicted to bind to a protein encoded by an HLA allele different from the second mutant GATA3 peptide sequence.

[0044] In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM. In some embodiments, at least one polypeptide comprises at least one mutant GATA3 peptide sequence that stably binds to a protein encoded by an HLA allele for more than 24 hours, more than 12 hours, more than 9 hours, more than 6 hours, more than 5 hours, more than 4 hours, more than 3 hours, more than 2 hours, more than 1 hour, more than 45 minutes, more than 30 minutes, more than 15 minutes, or more than 10 minutes. In some embodiments, the HLA allele is selected from the group consisting of HLA-A02:01, HLA-A24:02, HLA-A03:01, HLA-B07:02, HLA-B08:01, and any combination thereof.

[0045] In some embodiments, at least one polypeptide comprises at least one of the following sequences: TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI; and / or MFLKAESKI and / or YMFLKAESKI, VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR; and / or FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL and / or IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI.

[0046] In some embodiments, the two or more mutant GATA3 peptide sequences comprise at least two of the following sequences: TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI; and / or MFLKAESKI and / or YMFLKAESKI, VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR; and / or FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL and / or IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI.

[0047] In some embodiments, the mutant GATA3 peptide sequence comprises at least two of the following sequences: EPCSMLTGPPARVPAVPFDLH, SMLTGPPARVPAVPFDLH, GPPARVPAVPFDLHFCRSSIMKPKRD, DLHFCRSSIMKPKRDGYMFLKAESKI, KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH, FLKAESKIMFATLQRS, and KPKRDGYMFLKAESKI.

[0048] In some embodiments, the mutant GATA3 peptide sequence comprises at least two sequences in Table 5 and / or Table 6. In some embodiments, a first mutant GATA3 peptide sequence of the two or more mutant GATA3 peptide sequences comprises a first neoepitope of a GATA3 protein and a second mutant GATA3 peptide sequence comprises a second neoepitope of a mutant GATA protein, wherein the first mutant GATA3 peptide sequence is different from the mutant GATA3 peptide sequence, the first neoepitope comprises at least one mutant amino acid, and the second neoepitope comprises the same mutant amino acid.

[0049] In embodiments, a composition comprising at least one polypeptide comprising one or more mutant GATA3 peptide sequences, wherein the at least one polypeptide comprises:

[0050] [Xaa] F -[Xaa] N -[Xaa] C wherein each Xaa is independently any amino acid, [Xaa] N -[Xaa] C represents one or more mutant GATA3 peptide sequences, and [Xaa] N and [Xaa] C Each contains a contiguous amino acid sequence encoded by a different portion of the GATA3 gene, [Xaa] N is encoded by a non-wild-type reading frame and contains [Xaa] C comprises at least one mutant amino acid and is encoded in a non-wild-type reading frame, N is an integer between 0 and 100, C is an integer between 1 and 100, F is an integer between 0 and 100, and the sum of N and M is at least 8.

[0051] In some embodiments, for each of the mutant GATA3 peptide sequences, at least 8 consecutive amino acids are [Xaa] F -[Xaa] N -[Xaa]C or [Xaa] N -[Xaa] C -[Xaa] F wherein each Xaa is an amino acid, [Xaa] N and [Xaa] C Each contains an amino acid sequence encoded by a different portion of the GATA3 gene, [Xaa] F is any amino acid sequence, [Xaa] N is encoded in a non-wild-type reading frame of the GATA3 gene and is [Xaa] C contains at least one mutant amino acid and is encoded in a non-wild-type reading frame of the GATA3 gene, N is an integer of 0 to 100, C is an integer of 1 to 100, F is an integer of 0 to 100, and the sum of N and M is at least 8. In some embodiments, [Xaa] F each Xaa is a lysine residue, and F is an integer from 1 to 100, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, F is 3, 4, or 5.

[0052] In some embodiments, the at least one mutant amino acid comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive mutant amino acids. In some embodiments, each of the mutant GATA3 peptide sequences is present at a concentration of at least 1 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 200 μg / mL, at least 250 μg / mL, at least 300 μg / mL, or at least 400 μg / mL. In some embodiments, each of the mutant GATA3 peptide sequences is present only at a concentration of at most 5000 μg / mL, at most 2500 μg / mL, at most 1000 μg / mL, at most 750 μg / mL, at most 500 μg / mL, at most 400 μg / mL, or at most 300 μg / mL. In some embodiments, each of the mutant GATA3 peptide sequences is present at a concentration of 10 μg / mL to 5000 μg / mL, 10 μg / mL to 4000 μg / mL, 10 μg / mL to 3000 μg / mL, 10 μg / mL to 2000 μg / mL, 10 μg / mL to 1000 μg / mL, 25 μg / mL to 500 μg / mL, 50 μg / mL to 500 μg / mL, 100 μg / mL to 500 μg / mL, 200 μg / mL to 500 μg / mL, 200 μg / mL to 400 μg / mL, or 3000 μg / mL to 400 μg / mL.

[0053] In some embodiments, the composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the adjuvant is poly-ICLC. In an aspect, provided herein is a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a pH adjusting agent present at a concentration of less than 1 mM or more than 1 mM. In some embodiments, the pharmaceutical composition is a vaccine composition. In some embodiments, the pharmaceutical composition is aqueous.

[0054] In some embodiments, one or more of the at least one polypeptide are limited by pI > 5 and HYDRO > -6, pI > 8 and HYDRO > -8, pI < 5 and HYDRO > -5, pI > 9 and HYDRO < -8, pI > 7 and a HYDRO value > -5.5, pI < 4.3 and -4 > HYDRO > -8, pI > 0 and HYDRO < -8, pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -8, pI > 0 and HYDRO > -4, or pI > 4.3 and HYDRO < -4, pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -9, 5 > pi > 12 and -4 > HYDRO > -9.

[0055] In some embodiments, the pH adjusting agent is a base. In some embodiments, the pH adjusting agent is a conjugate base of a weak acid. In some embodiments, the pH adjusting agent is a pharmaceutically acceptable salt. In some embodiments, the pH adjusting agent is a dicarboxylate or tricarboxylate. In some embodiments, the pH adjusting agent is citric acid and / or a citrate salt. In some embodiments, the citrate salt is disodium citrate and / or trisodium citrate. In some embodiments, the pH adjusting agent is succinic acid and / or a succinate salt. In some embodiments, the succinate salt is disodium succinate and / or monosodium succinate. In some embodiments, the succinate salt is disodium succinate hexahydrate. In some embodiments, the pH adjusting agent is present at a concentration of 0.1 mM to 10 mM. In some embodiments, the pH adjusting agent is present at a concentration of 0.1 mM to 5 mM. In some embodiments, the pH adjusting agent is present at a concentration of 0.1 mM to 1 mM. In some embodiments, the pH adjusting agent is present at a concentration of 1 mM to 10 mM. In some embodiments, the pH adjusting agent is present at a concentration of 1 mM to 5 mM.

[0056] In some embodiments, the pharmaceutically acceptable carrier comprises a liquid. In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises a sugar. In some embodiments, the sugar comprises dextrose or mannitol. In some embodiments, the dextrose or mannitol is present at a concentration of 1-10% w / v. In some embodiments, the sugar comprises trehalose. In some embodiments, the sugar comprises sucrose. In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO).

[0057] In some embodiments, DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, 1% to 5%, 2% to 5%, 2% to 4%, or 2% to 4%. In some embodiments, the pharmaceutically acceptable carrier does not include dimethyl sulfoxide (DMSO). In some embodiments, the pharmaceutical composition may be lyophilized. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator or adjuvant is selected from the group consisting of poly ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonist, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune (Juvlmmune), LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 Stimulon.

[0058] In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is 2:1 to 1:10 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:1, 1:1.5, 1:2, 1:3, 1:4, or 1:5 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:3 v:v.

[0059] In an aspect, provided herein is a method for synthesizing a GATA3 peptide, the method comprising the steps of: coupling at least one dipeptide or a derivative thereof with an amino acid or a derivative thereof of the GATA3 peptide or its derivative to obtain a pseudoproline containing GATA3 peptide or its derivative, wherein the dipeptide or its derivative comprises a pseudoproline moiety; coupling one or more selected amino acids, small peptides or derivatives thereof with the pseudoproline containing GATA3 peptide or its derivative; and cleaving the pseudoproline containing GATA3 peptide or its derivative from the resin.

[0060] In some embodiments, the method further comprises deprotecting the pseudoproline-containing GATA3 peptide or its derivative. In some embodiments, the GATA3 peptide is a peptide of at least one polypeptide of the compositions described herein or the pharmaceutical compositions herein. In some embodiments, the N-terminal amino acid or its derivative of the GATA3 peptide or its derivative is bound to a resin. In some embodiments, the resin is a Wang resin or a 2-chlorotrityl resin (2-Cl-Trt resin). In some embodiments, the starting material for coupling is Fmoc-His(Trt)-Wang resin, H-His(Trt)-2Cl-Trt resin, Fmoc-Asp(OtBu)-Wang resin, Fmoc-Ile-Wang resin, Fmoc-Ser(tBu)-Wang resin, or Fmoc-Leu-Wang resin. In some embodiments, the amino acid or derivative thereof to which the at least one dipeptide or derivative thereof is coupled is selected from the group consisting of Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, Tyr, His, and Val.

[0061] In some embodiments, one or more selected amino acids, small peptides or derivatives thereof optionally coupled to a pseudoproline containing GATA3 peptide or derivative thereof may be selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc- Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(t Bu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH.

[0062] In some embodiments, the N-terminal amino acid or derivative thereof of the GATA3 peptide or derivative thereof is selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc c-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH.

[0063] In some embodiments, the pseudoproline moiety is Fmoc-Ser(tBu)-Ser(psi(Me,Me)pro)-OH. In some embodiments, the pseudoproline moiety is Fmoc-Ala-Thr(psi(Me,Me)pro)-OH. In some embodiments, the pseudoproline moiety is Fmoc-Glu(OtBu)-Ser(psi(Me,Me)pro)-OH. In some embodiments, the pseudoproline moiety is Fmoc-Leu-Thr(psi(Me,Me)pro)-OH. In some embodiments, the pseudoproline moiety is Fmoc-Leu-Cys(psi(Dmp,H)pro)-OH.

[0064] In some embodiments, Xaa-Ser is Ser-Ser. In some embodiments, Xaa-Ser is Glu-Ser. In some embodiments, Xaa-Thr is Ala-Thr. In some embodiments, Xaa-Thr is Leu-Thr. In some embodiments, Xaa-Cys is Leu-Cys.

[0065] In an aspect, provided herein is a method of treating a subject having cancer, the method comprising administering to the subject a pharmaceutical composition described herein.

[0066] In an embodiment, a method of identifying a subject having cancer as a candidate for a therapeutic agent includes identifying the subject as expressing a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele; and wherein the therapeutic agent is at least one polypeptide comprising one or more mutant GATA3 peptide sequences, each of the one or more mutant GATA3 peptide sequences comprising at least one mutant amino acid and being a fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in a GATA3 gene in the cancer cell. Provided herein are methods comprising at least one polypeptide; comprising at least one polynucleotide comprising a sequence encoding at least one polypeptide; comprising one or more APCs comprising at least one polypeptide; or comprising a T cell receptor (TCR) specific for a neoepitope of at least one polypeptide in a complex with an HLA protein; wherein each of the one or more mutant GATA3 peptide sequences or a portion thereof binds to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele. In some embodiments, the method further comprises administering a therapeutic agent to the subject.

[0067] In an embodiment, a method of treating a subject having cancer comprises administering a composition to the subject; the composition comprises at least one polypeptide comprising one or more mutant GATA3 peptide sequences, each of the one or more mutant GATA3 peptide sequences comprising at least one mutant amino acid and being a fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in a GATA3 gene in a cancer cell; at least one polynucleotide comprising a sequence encoding the at least one polypeptide; or one or more polypeptides comprising the at least one polypeptide. or a T cell receptor (TCR) specific for a neoepitope of at least one polypeptide, wherein the mutant GATA3 peptide or a portion thereof binds to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele; and the subject is identified as expressing an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele.

[0068] In an aspect, provided herein is a method of treating a subject having cancer, comprising administering a composition to the subject, wherein the composition comprises at least one polypeptide comprising two or more mutant GATA3 peptide sequences, each of the two or more mutant GATA3 peptide sequences comprising at least 8 consecutive amino acids of SEQ ID NO: 1, and wherein the C-terminus of the first GATA3 peptide sequence overlaps with the N-terminus of the second GATA3 peptide sequence; at least one polynucleotide comprising a sequence encoding the at least one polypeptide; one or more APCs comprising the at least one polypeptide; or a T cell receptor (TCR) specific to a neoepitope of the at least one polypeptide, the T cell receptor (TCR) forming a complex with an HLA protein, wherein the HLA allele expressed by the subject is unknown at the time of administration.

[0069] In some embodiments, an immune response is elicited in the subject. In some embodiments, the immune response is a humoral response. In some embodiments, the mutant GATA3 peptide sequence is administered simultaneously, separately, or sequentially. In some embodiments, the first peptide is administered sequentially after a period sufficient for the second peptide to activate the second T cell. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL). In some embodiments, the subject has breast cancer that is resistant to anti-estrogen therapy, is MSI breast cancer, is metastatic breast cancer, is Her2-negative breast cancer, is Her2-positive breast cancer, is ER-negative breast cancer, is ER-positive breast cancer, is PR-positive breast cancer, is PR-negative breast cancer, or any combination thereof. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the method further comprises administering at least one additional therapeutic agent or therapy.

[0070] In some embodiments, the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 and anti-PD-L1 agent, an anti-CTLA-4 agent, an anti-CD40 agent, letrozole, fulvestrant, and / or a CDK4 / 6 inhibitor. In some embodiments, the at least one additional therapeutic agent is palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; alcyaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-aminothioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (lyubidin); and flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atubeciclib; bliciclib; ribiciclib; trilaciclib (G1T28); and any combination thereof.

[0071] In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after the administration of the mutant GATA3 peptide sequence. In some embodiments, the administering step comprises subcutaneous administration or intravenous administration. In some embodiments, the cancer is recurrent or metastatic breast cancer. In some embodiments, the subject has experienced disease progression after endocrine therapy in combination with a CDK4 / 6 inhibitor.

[0072] A common mutation in CLL and certain lymphomas is a cysteine ​​to serine change at position 481 (C481S) in the BTK (Bruton's tyrosine kinase) gene. The mutation occurs at the amino acid sequence: [ka] The mutated serine is underlined within the region having the nucleotide sequence ##STR00002## This change results in multiple binding peptides that bind to a variety of HLA molecules.

[0073] In one aspect, provided herein is a composition comprising a polypeptide comprising one or more mutant BTK peptide sequences from a C481S mutant BTK protein, wherein the one or more mutant BTK peptide sequences comprise at least 8 contiguous amino acids of the mutant BTK protein, and the amino acid sequence of the peptide is listed in Table 34: ANGSLLNY; ANGSLLNYL; ANGSLLNYLR; EYMANGSL; EYMANGSLLN; EYMANGSLLNY; GSLLNYLR; GSLLNYLREM; ITEYMANGS; ITEYMANGSL; ITEYMANGSLL; MANGOSLNYL; MANGOSLNYLR; NGSLLNYL; NGSLLNYL; SLLNYLREMR; TEYMANGSLL; TEYMANGSLLNY; YMANGSLL; or YMANGSLLN.

[0074] In some embodiments, the one or more mutant BTK peptide sequences (a) comprise ANGSLLNY and bind or are predicted to bind to a protein encoded by the HLA-A36:01 allele, or (b) comprise ANGSLLNYL and bind or are predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-C15:02, HLA-C08:01, HLA-C06:02, HLA-A02:04, HLA-C12:02, HLA-B44:02, HLA-C17:01, and HLA-B38:01. (c) comprises ANGSLLNYLR and binds or is predicted to bind to a protein encoded by an HLA-A74:01 allele or an HLA-A31:01 allele; (d) comprises EYMANGSL and binds or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-C14:02, HLA-C14:03, and HLA-A24:02; or (e) comprises EYMANGSLLN and binds or is predicted to bind to a protein encoded by an HLA-A24:02 allele or an HLA-A23:01 allele. (f) comprises EYMANGSLLNY and binds or is predicted to bind to a protein encoded by the HLA-A29:02 allele; (g) comprises GSLLNYLR and binds or is predicted to bind to a protein encoded by the HLA-A31:01 allele or the HLA-A74:01 allele; or (h) comprises GSLLNYLREM and binds to a protein encoded by the HLA-B58:02 allele or the HLA-B57:01 allele; or (i) comprises ITEYMANGS and binds or is predicted to bind to a protein encoded by the HLA-A01:01 allele; (j) comprises ITEYMANGSL and binds or is predicted to bind to a protein encoded by the HLA-A01:01 allele; (k) comprises ITEYMANGSLL and binds or is predicted to bind to a protein encoded by the HLA-A01:01 allele; (l) comprises MANGSLLNYL and binds to HLA-C17:01, HLA-C02:02,(m) a polypeptide that binds to or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-B35:01, HLA-C03:03, HLA-C08:01, HLA-B35:03, HLA-C12:02, HLA-C01:02, HLA-C03:04, and HLA-C08:02, including MANGSLLNYLR, and that binds to or is predicted to bind to a protein encoded by the HLA-A33:03 allele or the HLA-A74:01 allele, or (n) a polypeptide that binds to or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-B35:01, HLA-C03:03, HLA-C08:01, HLA-B35:03, HLA-C12:02, HLA-C01:02, HLA-C03:04, and HLA-C08:02, including MANGSLLNYLR, and that binds to or is predicted to bind to a protein encoded by the HLA-A33:03 allele or the HLA-A74:01 allele, or YL and binds or is predicted to bind to a protein encoded by the HLA-B14:02 allele; (o) NGSLLNYL and binds or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-A68:01, HLA-A33:03, HLA-A31:01 and HLA-A74:01; or (p) SLLNYLREMR and binds to a protein encoded by the HLA-A74:01 allele or the HLA-A31:01 allele. (q) comprises TEYMANGSLL and binds or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-B40:01, HLA-B44:03, HLA-B49:01, HLA-B44:02 and HLA-B40:02; (r) comprises TEYMANGSLLNY and binds or is predicted to bind to a protein encoded by the HLA-B44:03 allele; or (s) comprises YMANGSLL and binds to HLA-B15:09 , (t) binds to or is predicted to bind to a protein encoded by an HLA allele selected from the group consisting of HLA-C03:04, HLA-C03:03, HLA-C17:01, HLA-C03:02, HLA-C14:03, HLA-C14:02, HLA-C04:01, HLA-C02:02, and HLA-A01:01, or (t) contains YMANGSLLN and binds to or is predicted to bind to a protein encoded by the HLA-A29:02 allele or the HLA-A01:01 allele.

[0075] In some embodiments, one or more mutant BTK peptide sequences are specific for a cognate T cell receptor in complex with an HLA protein, hi some embodiments, a composition comprises two or more mutant BTK peptide sequences.

[0076] In one aspect, provided herein is a composition comprising at least one polypeptide comprising one or more mutant BTK peptide sequences selected from Table 34, each having at least 8 contiguous amino acids from a C481S mutant BTK protein, the composition further comprising three or more amino acid residues heterologous to the mutant BTK protein linked to the N- or C-terminus of the mutant BTK peptide sequence, the three or more amino acid residues enhancing intracellular processing of the mutant BTK peptide sequence and / or enhancing presentation of an epitope of the mutant BTK peptide sequence. In some embodiments, the three or more amino acid residues heterologous to the mutant BTK protein comprise amino acid sequences from CMV-pp65, HIV, MART-1, or a non-viral, non-BTK endogenous peptide.

[0077] In some embodiments, the three or more amino acid residues that are heterologous to the mutant BTK protein include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0078] In some embodiments, the three or more amino acid residues that are heterologous to the mutant BTK protein include at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 amino acids.

[0079] In one aspect, the formula (N-terminal Xaa) N -(Xaa BTK ) P -(Xaa-C terminus) C (wherein P is an integer greater than 7; and (Xaa BTK ) P is a mutant BTK peptide sequence comprising at least 8 consecutive amino acids selected from the sequence of a mutant BTK protein comprising the C481S mutant amino acid, IFIITEYMANGSLLNYLREMRHR; N is (i) 0 or (ii) an integer greater than 2; (N-terminal Xaa) N is any amino acid sequence heterologous to the mutant BTK protein; C is (i) 0 or (ii) an integer greater than 2; (Xaa-C-terminus) C is any amino acid sequence heterologous to the mutant BTK protein; and N and C are both 0.

[0080] In some embodiments, (N-terminal Xaa) N and / or (Xaa-C terminus) C comprises the amino acid sequence of a CMV-pp65, HIV, MART-1, or non-viral, non-BTK endogenous protein or peptide.

[0081] In some embodiments, N and / or C are integers greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0082] In some embodiments, N and / or C are integers less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100. In some embodiments, the composition of any one of claims 8-10, wherein N is 0. In some embodiments, C is 0.

[0083] In one aspect, provided herein is a composition comprising a polynucleotide sequence encoding the polypeptide of claim 1. In one aspect, the composition comprises a polynucleotide sequence encoding one or more peptide sequences for any of the mutant BTK peptides listed above and in Tables 34 and 36. In some embodiments, at least one polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 mutant BTK peptide sequences. In some embodiments, at least one of the mutant BTK peptide sequences comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous amino acids of a mutant BTK protein. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutant BTK peptide sequences comprise at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of the mutant BTK protein. In some embodiments, each of the mutant BTK peptide sequences, or each of the two or more BTK peptide sequences, comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous amino acids of a mutant BTK protein. In some embodiments, at least one polypeptide comprises at least one mutant BTK peptide sequence that binds, or is predicted to bind, to a protein encoded by an HLA allele listed in Table 35 with an affinity of 150 nM or less and / or a half-life of 2 hours or longer.In some embodiments, the mutant BTK peptide sequence comprises: (a) a first mutant BTK peptide sequence selected from Table 34, wherein the first mutant BTK peptide sequence binds to or is predicted to bind to a protein encoded by the HLA allele; and (b) a second BTK peptide having a C481S mutation, wherein the first mutant BTK peptide sequence and the second mutant BTK peptide sequence are non-identical.

[0084] In some embodiments, at least one polypeptide comprises at least one mutant BTK peptide sequence that binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM.

[0085] In some embodiments, at least one polypeptide comprises at least one mutant BTK peptide sequence that stably binds to a protein encoded by an HLA allele for more than 24 hours, more than 12 hours, more than 9 hours, more than 6 hours, more than 5 hours, more than 4 hours, more than 3 hours, more than 2 hours, more than 1 hour, more than 45 minutes, more than 30 minutes, more than 15 minutes, or more than 10 minutes.

[0086] In some embodiments, (N-terminal Xaa) N comprises the amino acid sequence of IDIIMKIRNA, FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC, FFFFFFFFFFFFFFFFFFFFFFFFFFAAFWFW, IFFIFFIIFFFFFFFFFFFFIIIIIIIWEC, FIFFFIIFFFFFFFFFFFIFIFIIIFWEC, TEY, WQAGILAR, HSYTTAE, PLTEEKIK, GALHFKPGSR, RRANKDATAE, KAFISHEEKR, TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK, or MTEYKLVVV. Ccomprises the amino acid sequence of KKNKKDDIKD, AGNDDDDDDDDDDDDDDDDDKKDKDDDDDD, AGNKKKKKKKNNNNNNNNNNNNNNNNNNNN, AGRDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD, GKSALTIQL, GKSALTI, QGQNLKYQ, ILGVLLLI, EKEGKISK, AASDFIFLVT, KELKQVASPF, KKKLINEKKE, KKCDISLQFF, KSTAGDTHLG, ATFYVAVTVP, LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG, or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE.

[0087] In some embodiments, at least one of the mutant BTK peptide sequences comprises a mutant amino acid that is not encoded by the genome of the subject's cancer cell.

[0088] In some embodiments, each of the mutant BTK peptide sequences is present at a concentration of at least 1 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL. In some embodiments, each of the mutant BTK peptide sequences is present at a concentration of at most 5000 μg / mL, at most 2500 μg / mL, at most 1000 μg / mL, at most 750 μg / mL, at most 500 μg / mL, at most 400 μg / mL, or at most 300 μg / mL. In some embodiments, each of the mutant BTK peptide sequences is present at a concentration of 10 μg / mL to 5000 μg / mL, 10 μg / mL to 4000 μg / mL, 10 μg / mL to 3000 μg / mL, 10 μg / mL to 2000 μg / mL, 10 μg / mL to 1000 μg / mL, 25 μg / mL to 500 μg / mL, or 50 μg / mL to 300 μg / mL. In some embodiments, the composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the adjuvant is poly-ICLC.

[0089] In one aspect, provided herein is a pharmaceutical composition comprising (a) the composition described above and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises a pH adjuster. In some embodiments, the pharmaceutical composition is a vaccine composition. In some embodiments, the pharmaceutical composition is aqueous. In some embodiments, the pharmaceutical composition comprises one or more of at least one polypeptide limited by: (a) pI > 5 and HYDRO > -6; (b) pI > 8 and HYDRO > -8; (c) pI < 5 and HYDRO > -5; (d) pI > 9 and HYDRO < -8; (e) pI > 7 and a HYDRO value > -5.5; (f) pI < 4.3 and -4 > HYDRO > -8; (g) pI > 0 and HYDRO < -8, pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -8; (h) pI > 0 and HYDRO > -4, or pI > 4.3 and HYDRO < -4; (i) pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -9; (j) 5 > pi > 12 and -4 > HYDRO > -9.

[0090] In some embodiments, the pH adjusting agent is a base. In some embodiments, the pH adjusting agent is a conjugate base of a weak acid. In some embodiments, the pH adjusting agent is a pharmaceutically acceptable salt. In some embodiments, the pH adjusting agent is a dicarboxylate or tricarboxylate. In some embodiments, the pH adjusting agent is citric acid and / or a citrate salt. In some embodiments, the citrate salt is disodium citrate and / or trisodium citrate. In some embodiments, the pH adjusting agent is succinic acid and / or a succinate salt. In some embodiments, the succinate salt is disodium succinate and / or monosodium succinate. In some embodiments, the succinate salt is disodium succinate hexahydrate. In some embodiments, the pH adjusting agent is present at a concentration of 0.1 mM to 1 mM. In some embodiments, the pharmaceutically acceptable carrier comprises a liquid. In some embodiments, the pharmaceutically acceptable carrier comprises water.

[0091] In some embodiments, the pharmaceutically acceptable carrier comprises a sugar. In some embodiments, the sugar comprises dextrose. In some embodiments, the dextrose is present at a concentration of 1-10% w / v. In some embodiments, the sugar comprises trehalose. In some embodiments, the sugar comprises sucrose.

[0092] In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO). In some embodiments, DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%. In some embodiments, the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide (DMSO). In some embodiments, the pharmaceutical composition may be lyophilized. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some embodiments, the immunomodulator or adjuvant is selected from the group consisting of poly ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonist, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 Stimulon.

[0093] In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is 2:1 to 1:10 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:1, 1:2, 1:3, 1:4, or 1:5 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:3 v:v.

[0094] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject the pharmaceutical composition described above.

[0095] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising a peptide having a sequence selected from the left column of Table 34, 36, or 37, wherein the subject expresses a protein encoded by any one of the HLA alleles listed in the right column corresponding to the peptide in the table. In some embodiments, the present invention provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a composition comprising one or more mutant BTK peptides or a composition comprising one or more nucleic acids encoding one or more mutant BTK peptides, wherein each mutant BTK peptide comprises at least 8 contiguous amino acids of a mutant BTK protein comprising the C481S mutation, and wherein at least one of the one or more peptides binds to a protein encoded by an HLA allele listed in Table 34, 36, or 37 expressed by the subject. In some embodiments, the peptide binds to an HLA protein with an affinity of 150 nM or less and / or a half-life of 2 hours or longer.

[0096]

[0013] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a first and a second peptide, or a nucleic acid encoding the first and second peptides, wherein the first peptide has an amino acid sequence selected from Table 34, 36, or 37, and the second peptide has an amino acid sequence selected from any one of Tables 34, 36, or 37.

[0097] In some embodiments, an immune response is elicited in the subject. In some embodiments, the immune response is a humoral response.

[0098] In some embodiments, one or more mutant BTK peptides are administered simultaneously, separately, or sequentially.

[0099] In some embodiments, the second peptide is administered sequentially after a period sufficient for the first peptide to activate the second T cells.

[0100] In some embodiments, the cancer is selected from the group consisting of certain types of lymphoma and certain types of leukemia, hi some embodiments, the cancer is acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), chronic lymphocytic lymphoma, or B-cell non-Hodgkin's lymphoma.

[0101] In some embodiments, the method further comprises administering at least one additional therapeutic agent or therapy.

[0102] In some embodiments, the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof.

[0103] In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 and anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered prior to, concurrently with, or following administration of the mutant BTK peptide sequence.

[0104] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising: (a) identifying a first protein expressed by the subject, wherein the first protein is encoded by a first HLA allele in the subject, wherein the first HLA allele is an HLA allele provided in any one of Tables 34, 37, or 38; (b) administering to the subject (i) a first mutant BTK peptide that is a peptide against the first HLA allele provided according to any one of Tables 34, 36, or 37, or (ii) a polynucleic acid encoding the first mutant BTK peptide.

[0105] In one aspect, provided herein is a method of identifying a subject having cancer as a candidate for a therapeutic agent, comprising the step of identifying the subject as a subject who expresses a protein encoded by an HLA-identified in one of Tables 34, 36, or 37; wherein the therapeutic agent is a mutant BTK peptide or a nucleic acid encoding a mutant BTK peptide, wherein the mutant BTK peptide comprises at least 8 contiguous amino acids of a mutant BTK protein comprising a mutation at C481, wherein the peptide (i) comprises a C481S mutation, (ii) comprises the sequence of a peptide in any one of Tables 34, 36, or 37, and (iii) binds to a corresponding protein encoded by an HLA-identified in any one of Tables 34, 36, or 37.

[0106] In some aspects, provided herein are compositions comprising a polypeptide comprising one or more mutant EGFR peptide sequences from a T790M mutant EGFR protein, wherein the one or more mutant EGFR peptide sequences comprise at least 8 contiguous amino acids selected from the group consisting of LIMQLMPF, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM, VQLIMQLM, STVQLIMQL, and LTSTVQLIM.

[0107] In some embodiments, the one or more mutant EGFR peptide sequences are specific for a cognate T cell receptor in complex with an HLA protein.

[0108] In some embodiments, the composition comprises a mixture of two or three or more mutant EGFR peptide sequences. In some embodiments, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutant EGFR peptide sequences. In some embodiments, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids of a mutant EGFR protein.

[0109] In some aspects, provided herein are compositions comprising at least one polypeptide comprising one or more mutant EGFR peptide sequences from a T790M mutant EGFR protein, wherein the one or more mutant EGFR peptide sequences comprise at least 8 contiguous amino acids selected from the group consisting of LIMQLMPF, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM, VQLIMQLM, STVQLIMQL and LTSTVQLIM, further comprising three or more amino acid residues heterologous to the mutant EGFR protein linked to the N-terminus or C-terminus of the mutant EGFR peptide sequence, wherein the three or more amino acid residues enhance processing of the mutant EGFR peptide sequence in cells and / or enhance presentation of an epitope of the mutant EGFR peptide sequence.

[0110] In some embodiments, the three or more amino acid residues that are heterologous to the mutant EGFR protein comprise amino acid sequences from CMV-pp65, HIV, MART-1, or a non-viral, non-EGFR endogenous peptide.

[0111] In some embodiments, the three or more amino acid residues that are heterologous to the mutant EGFR protein include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids.

[0112] In some embodiments, the three or more amino acid residues heterologous to the mutant EGFR protein that are linked to the N-terminus or C-terminus of the two or more mutant EGFR peptide sequences comprise at most only 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90 or 100 amino acids.

[0113] In some embodiments, (Xaa-C-terminus) C is any amino acid sequence heterologous to the mutant EGFR protein, where both N and C are not zero.

[0114] In some embodiments, (N-terminal Xaa) N and / or (Xaa-C terminus) C comprises the amino acid sequence of CMV-pp65, HIV, MART-1 or a non-viral, non-EGFR endogenous protein or peptide.

[0115] In some embodiments, N and / or C are integers greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.

[0116] In some embodiments, N and / or C are integers less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100. In some embodiments, n is 0. In some embodiments, C is 0.

[0117] In one aspect, provided herein is a composition comprising a polynucleotide sequence encoding the above polypeptide. In one embodiment, the composition comprises a polynucleotide sequence encoding one or more mutant EGFR peptide sequences disclosed herein.

[0118] In some embodiments, the composition comprising one or more mutant EGFR peptide sequences further comprises one or more mutant EGFR peptides selected from Tables 40A-40D.

[0119] In some embodiments, at least one polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 mutant EGFR peptide sequences.

[0120] In some embodiments, at least one of the mutant EGFR peptide sequences comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of a mutant EGFR protein. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the mutant EGFR peptide sequences comprise at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of the mutant EGFR protein. In some embodiments, each of the mutant EGFR peptide sequences, or each of the two or more EGFR peptide sequences, comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids of a mutant EGFR protein.

[0121] In some embodiments, at least one polypeptide comprises at least one mutant EGFR peptide sequence that binds, or is predicted to bind, to a protein encoded by an HLA allele listed in Table 41 with an affinity of 150 nM or less and / or a half-life of 2 hours or longer.

[0122] In some embodiments, the mutant EGFR peptide sequence comprises a first mutant EGFR peptide sequence selected from the group consisting of STVQLIMQL, LIMQLMPF, LTSTVQLIM, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM, and VQLIMQLM, and a second mutant EGFR peptide sequence having a T790M mutation.

[0123] In some embodiments, the mutant EGFR peptide sequence is (a) a first mutant EGFR peptide sequence selected from the group consisting of STVQLIMQL, LIMQLMPF, LTSTVQLIM, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM, and VQLIMQLM, and is selected from the group consisting of HLA-A68:02, HLA-C15:02, HLA-A25:01, HLA-B 57:03, HLA-C12:02, HLA-C03:02, HLA-A26:01, HLA-C12:03, HLA-C06:02, HLA-C03:03, HLA-B52:01, HLA- A30:01, HLA-C02:02, HLA-C12:03, HLA-A11:01, HLA-A32:01, HLA-A02:04, HLA-A68:01, HLA-B15:09, HLA -C17:01, HLA-C03:04, HLA-B08:01, HLA-A01:01, HLA-B42:01, HLA-B57:01, HLA-B15:01, HLA-B14:02, HL A-B37:01, HLA-A36:01, HLA-C15:02, HLA-B15:09, HLA-C12:02, HLA-B38:01, HLA-C03:03, HLA-A02:03, H and (b) a first mutant EGFR peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-B58:02, HLA-C08:01, HLA-B35:01, HLA-B40:01, and / or HLA-B35:03 allele, and (b) a second mutant EGFR peptide sequence having a T790M mutation, wherein the first and second peptides are not identical.

[0124] In some embodiments, at least one polypeptide comprises at least one mutant EGFR peptide sequence that binds to a protein encoded by an HLA allele with an affinity of less than 10 μM, less than 1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, or less than 50 nM.

[0125] In some embodiments, at least one polypeptide comprises at least one mutant EGFR peptide sequence that stably binds to a protein encoded by an HLA allele for more than 24 hours, more than 12 hours, more than 9 hours, more than 6 hours, more than 5 hours, more than 4 hours, more than 3 hours, more than 2 hours, more than 1 hour, more than 45 minutes, more than 30 minutes, more than 15 minutes, or more than 10 minutes.

[0126] In some embodiments, (N-terminal Xaa) N comprises the amino acid sequence of IDIIMKIRNA, FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC, FFFFFFFFFFFFFFFFFFFFFFFFFFAAFWFW, IFFIFFIIFFFFFFFFFFFFFFIIIIIIIWEC, FIFFFIIFFFFFFFFFFFIFIIIIIIFWEC, TEY, WQAGILAR, HSYTTAE, PLTEEKIK, GALHFKPGSR, RRANKDATAE, KAFISHEEKR, TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK, or MTEYKLVVV.

[0127] In some embodiments, (C-terminal Xaa) C comprises the amino acid sequence of KKNKKDDIKD, AGNDDDDDDDDDDDDDDDDDKKDKDDDDDD, AGNKKKKKKKNNNNNNNNNNNNNNNNNNNN, AGRDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD, GKSALTIQL, GKSALTI, QGQNLKYQ, ILGVLLLI, EKEGKISK, AASDFIFLVT, KELKQVASPF, KKKLINEKKE, KKCDISLQFF, KSTAGDTHLG, ATFYVAVTVP, LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG, or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE.

[0128] In some embodiments, at least one of the mutant EGFR peptide sequences comprises a mutant amino acid that is not encoded by the genome of the subject's cancer cells.

[0129] In some embodiments, the mutant EGFR peptide sequence is present at a concentration of at least 1 μg / mL, at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, or at least 100 μg / mL.

[0130] In some embodiments, each of the mutant EGFR peptide sequences is present only at a concentration of at most 5000 μg / mL, at most 2500 μg / mL, at most 1000 μg / mL, at most 750 μg / mL, at most 500 μg / mL, at most 400 μg / mL, or at most 300 μg / mL.

[0131] In some embodiments, each of the mutant EGFR peptide sequences is present at a concentration of 10 μg / mL to 5000 μg / mL, 10 μg / mL to 4000 μg / mL, 10 μg / mL to 3000 μg / mL, 10 μg / mL to 2000 μg / mL, 10 μg / mL to 1000 μg / mL, 25 μg / mL to 500 μg / mL, or 50 μg / mL to 300 μg / mL.

[0132] In some embodiments, the composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the adjuvant is poly-ICLC.

[0133] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a composition comprising at least one polypeptide comprising at least one mutant EGFR peptide sequence as described above; and (b) a pharmaceutically acceptable excipient.

[0134] In some embodiments, the pharmaceutical composition further comprises a pH adjuster.

[0135] In some embodiments, the pharmaceutical composition is a vaccine composition.

[0136] In some embodiments, the pharmaceutical composition is aqueous.

[0137] In some embodiments, one or more of the at least one polypeptide are limited by pI > 5 and HYDRO > -6, pI > 8 and HYDRO > -8, pI < 5 and HYDRO > -5, pI > 9 and HYDRO < -8, pI > 7 and a HYDRO value > -5.5, pI < 4.3 and -4 > HYDRO > -8, pI > 0 and HYDRO < -8, pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -8, pI > 0 and HYDRO > -4, or pI > 4.3 and HYDRO < -4, pI > 0 and HYDRO > -4, or pI > 4.3 and -4 > HYDRO > -9, 5 > pi > 12 and -4 > HYDRO > -9.

[0138] In some embodiments, the pharmaceutical composition comprises a pH adjusting agent that is a base.

[0139] In some embodiments, the pH adjuster is a conjugate base of a weak acid.

[0140] In some embodiments, the pH adjusting agent is a pharmaceutically acceptable salt.

[0141] In some embodiments, the pH adjuster is a dicarboxylate or tricarboxylate.

[0142] In some embodiments, the pH adjuster is citric acid and / or citrate salts.

[0143] In some embodiments, the citrate salt is disodium citrate and / or trisodium citrate.

[0144] In some embodiments, the pH adjuster is succinic acid and / or a succinate salt.

[0145] In some embodiments, the succinate salt is disodium succinate and / or monosodium succinate.

[0146] In some embodiments, the succinate salt is disodium succinate hexahydrate.

[0147] In some embodiments, the pH adjuster is present at a concentration of 0.1 mM to 1 mM.

[0148] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, which comprises a liquid.

[0149] In some embodiments, the pharmaceutically acceptable carrier comprises water.

[0150] In some embodiments, the pharmaceutically acceptable carrier comprises a sugar.

[0151] In some embodiments, the sugar comprises dextrose.

[0152] In some embodiments, the dextrose is present at a concentration of 1-10% w / v.

[0153] In some embodiments, the sugar comprises trehalose.

[0154] In some embodiments, the sugar comprises sucrose.

[0155] In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO).

[0156] In some embodiments, DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%.

[0157] In some embodiments, the pharmaceutically acceptable carrier does not include dimethyl sulfoxide (DMSO).

[0158] In some embodiments, the pharmaceutical composition may be lyophilized.

[0159] In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant.

[0160] In some embodiments, the immunomodulator or adjuvant is selected from the group consisting of Poly ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 Stimulon.

[0161] In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is 2:1 to 1:10 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:1, 1:2, 1:3, 1:4, or 1:5 v:v. In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:3 v:v.

[0162] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject the pharmaceutical composition described above.

[0163]

[0013] In one aspect, a method of treating cancer in a subject comprises administering to a subject in need thereof one or more mutant EGFR peptides or one or more nucleic acids encoding the one or more mutant EGFR peptides; each mutant EGFR peptide comprises at least 8 contiguous amino acids of a mutant EGFR protein comprising the mutation T790M; and one or more mutant EGFR peptides selected from the group consisting of those listed in Tables 40A-40B. and wherein at least one of the one or more peptides is selected from the group consisting of HLA-A68:02, HLA-C15:02, HLA-A25:01, HLA-B57:03, HLA-C12:02, HLA-C03:02, HLA-A26:01, HLA-C12:03, HLA-C06:02, HLA-C03:03, HLA-B52:01, HLA-A30:01, HLA-C02:02, HLA-C12:03, HLA-A11:01, HLA-A32:01, HLA-A02:04, HLA-A68:01, HLA-B15:09, HLA-C17:01, HLA-C03:04, HLA-B08:01, HLA-A01:01, HLA-B42:01, HLA- B57:01, HLA-B15:01, HLA-B14:02, HLA-B37:01, HLA-A36:01, HLA-C15:02, HLA-B15:09, HLA-C12:02, HLA-B38:01, HLA-C03: Provided herein are methods, wherein the antibody binds to a protein encoded by an HLA-A02:03, HLA-B58:02, HLA-C08:01, HLA-B35:01, HLA-B40:01, and / or HLA-B35:03 allele with an affinity of 150 nM or less and / or a half-life of 2 hours or longer, and binds or is predicted to bind to a protein encoded by said allele; and said allele is expressed by the subject.

[0164] In one aspect, a method of treating a subject having cancer comprises administering to a subject in need thereof a polypeptide comprising a mutant EGFR peptide sequence or a polynucleotide encoding the mutant EGFR peptide, wherein (a) the mutant EGFR peptide has the sequence LIMQLMPF and the subject expresses a protein encoded by an HLA-C03:02 allele, or (b) the mutant EGFR peptide has the sequence LTSTVQLIM and the subject expresses a protein encoded by an HLA-C12:03, HLA-C15:02, HLA-B57:0 (c) the mutant EGFR peptide has the sequence QLIMQLMPF and the subject expresses a protein encoded by an HLA allele selected from the group consisting of HLA-B57:01, HLA-A36:01, HLA-C12:02, HLA-C03:03, and HLA-B58:02; or (d) the mutant EGFR peptide has the sequence STVQLIMQL and the subject expresses a protein encoded by an HLA-A68:02, HLA-C15:02, HLA-A25:01, HLA-B5 7:03, HLA-C12:02, HLA-A26:01, HLA-C12:03, HLA-C06:02, HLA-C03:03, HLA-A30:01, HLA-C02:02, HLA-A11:01, HLA-A32:01, HLA-A02:04, HLA-A68:01, HLA-B15:09, HLA-C03:04, HLA-B38:01, HLA-B57:01, HLA-A02:03, HLA-C08:01, HLA-B35:01, and HLA-B40:01. (e) the mutant EGFR peptide has the sequence STVQLIMQLM and the subject expresses a protein encoded by the HLA-B57:01 allele; (f) the mutant EGFR peptide has the sequence TSTVQLIMQL and the subject expresses a protein encoded by the HLA-C15:02 allele; or (g) the mutant EGFR peptide has the sequence TVQLIMQL and the subject expresses a protein encoded by any of HLA-C17:01, HLA-B08:01, HLA-B42:01, HLA-B14:02, HLA-B37:01,Provided herein are methods in which (i) the mutant EGFR peptide has the sequence TVQLIMQLM and the subject expresses a protein encoded by an HLA allele selected from the group consisting of HLA-B52:01, HLA-B14:02, and HLA-B37:01, (ii) the mutant EGFR peptide has the sequence TVQLIMQLM and the subject expresses a protein encoded by an HLA allele selected from the group consisting of HLA-B15:09, (iii) the mutant EGFR peptide has the sequence TVQLIMQLM and the subject expresses a protein encoded by an HLA allele selected from the group consisting of HLA-B35:03, (iv) the mutant EGFR peptide has the sequence VQLIMQLM and the subject expresses a protein encoded by an HLA allele selected from the group consisting of HLA-B52:01, HLA-B14:02, and HLA-B37:01,

[0165] In some embodiments, the method further comprises administering a second polypeptide composition comprising at least one mutant EGFR peptide, wherein the second mutant EGFR peptide is selected from Tables 40A-40D.

[0166] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising: (a) identifying a first protein expressed by the subject, wherein the first protein is encoded by a first HLA allele in the subject, wherein the first HLA allele is an HLA allele provided in any one of Tables 41-43; and (b) administering to the subject (i) a first mutant EGFR peptide that is a peptide to the first HLA allele provided according to any one of Tables 42Ai and ii, 42B, or 43, or (ii) a polynucleic acid encoding the first mutant EGFR peptide. In some embodiments, the method of treating cancer in a subject comprises identifying one or more specific HLA subtypes expressed in the subject; and administering to the subject a composition comprising one or more mutant EGFR peptides described herein such that the one or more peptides bind to at least one HLA subtype expressed by the subject with an affinity of 150 nM or less and / or a half-life of 2 hours or longer.

[0167] In one aspect, provided herein is a method of treating cancer in a subject, the method comprising: (a) identifying a subject who expresses a protein encoded by an HLA-B57:01 allele in the subject's genome; (b) administering to the subject a composition comprising a peptide having the sequence STVQLIMQLM. In one embodiment, the method comprises: (a) determining whether the subject expresses a protein encoded by an HLA-A26:01 allele in the subject's genome; (b) administering to the subject a composition comprising a peptide having the sequence QLIMQLMPF.

[0168] In some embodiments, an immune response is elicited in the subject. In one embodiment, the immune response is a humoral response.

[0169] In some embodiments, one or more mutant EGFR peptide sequences are administered simultaneously, separately, or sequentially, hi some embodiments, the second peptide is administered sequentially after a period sufficient for the first peptide to activate the second T cells.

[0170] In some embodiments, the cancer is selected from the group consisting of glioblastoma, lung adenocarcinoma, non-small cell lung cancer, lung squamous cell carcinoma, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, gastric cancer, breast cancer, colorectal cancer, endometrial cancer, and esophageal cancer.

[0171] In some embodiments, the method further comprises administering at least one additional therapeutic agent or therapy.

[0172] In some embodiments, the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after administration of the mutant EGFR peptide sequence.

[0173]

[0013] In one aspect, provided herein is a method of identifying a subject having cancer as a candidate for a therapeutic agent, comprising: identifying the subject as a subject who expresses a protein encoded by one of HLAs in Tables 41, 42Ai, 42Aii, 42B, or 43; the therapeutic agent is a mutant EGFR peptide or a nucleic acid encoding the mutant EGFR peptide; the mutant EGFR peptide comprises at least 8 consecutive amino acids of a mutant EGFR protein comprising a mutation at T790; and the peptide (i) comprises a T790M mutation, (ii) comprises the sequence of a peptide in any one of Tables 42Ai, 42Aii, 42B, 43, or 44, and (iii) binds to a corresponding protein encoded by any one of HLAs in Tables 42Ai, 42Aii, 42B, 43, or 44.

[0174] In one aspect, provided herein is a method for identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising determining that the subject expresses a protein encoded by an HLA-B57:01 allele, wherein the therapeutic agent comprises a mutant EGFR peptide having the amino acid sequence STVQLIMQLM.

[0175] In one aspect, provided herein is a method of identifying a subject as a candidate for a therapeutic agent, comprising determining that the subject expresses a protein encoded by the HLA-A26:01 allele, wherein the therapeutic agent comprises a mutant EGFR peptide having the amino acid sequence QLIMQLMPF.

[0176] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. The present invention provides, for example, the following items. (Item 1) A pharmaceutical composition comprising: (a) at least one polypeptide comprising a first mutant GATA3 peptide sequence and a second mutant GATA3 peptide sequence, or a pharmaceutically acceptable salt thereof; (i) each of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprises at least 8 consecutive amino acids of SEQ ID NO: 1; and (ii) the at least one polypeptide or a pharmaceutically acceptable salt thereof, wherein the C-terminal sequence of the first mutant GATA3 peptide sequence overlaps with the N-terminal sequence of the second mutant GATA3 peptide sequence, and at least eight consecutive amino acids of SEQ ID NO: 1 comprise at least one amino acid of the sequence PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); or (b) at least one polynucleotide comprising a sequence encoding said at least one polypeptide. (Item 2) 2. The pharmaceutical composition of item 1, wherein the first mutant GATA3 peptide sequence or the second mutant GATA3 peptide sequence comprises at least 8 consecutive amino acids of SEQ ID NO:2. (Item 3) 3. The pharmaceutical composition according to any one of items 1 to 2, wherein the first mutant GATA3 peptide sequence and the second mutant peptide sequence comprise at least 8 consecutive amino acids of SEQ ID NO: 2. (Item 4) 4. The pharmaceutical composition according to any one of items 2 to 3, wherein the at least 8 consecutive amino acids of SEQ ID NO: 2 comprise at least 8 consecutive amino acids of the sequence PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGL (SEQ ID NO: 3). (Item 5) 5. The pharmaceutical composition according to any one of items 2 to 4, wherein the at least 8 consecutive amino acids of SEQ ID NO: 2 comprise at least one amino acid of the sequence EPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 4). (Item 6) 6. The pharmaceutical composition according to any one of items 1 to 5, wherein at least one of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprises at least 14 mutant amino acids. (Item 7) 7. The pharmaceutical composition according to any one of items 1 to 6, wherein the at least one polypeptide comprises at least three mutant GATA3 peptide sequences. (Item 8) 8. The pharmaceutical composition according to any one of items 1 to 7, wherein the at least one polypeptide comprises at least two polypeptides. (Item 9) 9. The pharmaceutical composition according to any one of items 1 to 8, wherein the at least one polypeptide further comprises a third mutant GATA3 peptide sequence, the third mutant GATA3 peptide sequence comprising at least 8 consecutive amino acids of SEQ ID NO: 1, and the at least 8 consecutive amino acids of SEQ ID NO: 1 comprising at least one amino acid of the sequence of SEQ ID NO: 2. (Item 10) 10. The pharmaceutical composition of item 9, wherein the third GATA3 mutant peptide comprises at least 8 consecutive amino acids of SEQ ID NO:2. (Item 11) 11. The pharmaceutical composition of any one of items 1 to 10, wherein the at least one polypeptide comprises at least one mutant GATA3 peptide sequence that binds to or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele. (Item 12) the at least one polypeptide (a) HLA-A02:01 allele and HLA-A24:02 allele, (b) HLA-A02:01 allele and HLA-B08:01 allele; (c) HLA-A24:02 allele and HLA-B08:01 allele, or (d) HLA-A02:01 allele, HLA-A24:02 allele, and HLA-B08:01 allele 12. The pharmaceutical composition according to any one of items 1 to 11, comprising at least one mutant GATA3 peptide sequence that binds to or is predicted to bind to a protein encoded by (Item 13) (a) the first mutant GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele; (b) the second GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, or an HLA-B08:01 allele; and the first mutant GATA3 peptide sequence binds to, or is predicted to bind to, a protein encoded by an HLA allele different from the second mutant GATA3 peptide sequence. (Item 14) 14. The pharmaceutical composition according to any one of items 1 to 13, wherein at least one of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence binds to a protein encoded by an HLA allele with an affinity of less than 500 nM. (Item 15) 15. The pharmaceutical composition according to any one of items 1 to 14, wherein at least one of the first mutant GATA3 peptide sequence and the second mutant peptide sequence stably binds to a protein encoded by an HLA allele for more than 1 hour. (Item 16) The at least one polypeptide has the following sequence: (a) TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI; (b) MFLKAESKI and / or YMFLKAESKI (c) VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR; (d) FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL, and / or (e) IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI 16. The pharmaceutical composition according to any one of items 1 to 15, comprising at least one of: (Item 17) The at least one polypeptide has the following sequence: (a) TLQRSSLWCL, VLPEPHLAL, HVLPEPHLAL, ALQPLQPHA, AIQPVLWTT, APAIQPVLWTT, SMLTGPPARV, MLTGPPARV, and / or YMFLKAESKI; (b) MFLKAESKI and / or YMFLKAESKI; (c) VLWTTPPLQH, YMFLKAESK and / or KIMFATLQR; (d) FATLQRSSL, EPHLALQPL, QPVLWTTPPL, GPPARVPAV, MFATLQRSSL, KPKRDGYMF and / or KPKRDGYMFL, and / or (e) IMKPKRDGYM, MFATLQRSSL, FLKAESKIMF, LHFCRSSIM, EPHLALQPL, FATLQRSSL, ESKIMFATL, FLKAESKIM and / or YMFLKAESKI 17. The pharmaceutical composition according to any one of items 1 to 16, comprising at least two of: (Item 18) the mutant GATA3 peptide sequence is (a) a first mutant GATA3 peptide sequence from (a) and a second mutant GATA3 peptide sequence from (b); (b) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (c); (c) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (d); (d) the first mutant GATA3 peptide sequence from (a) and the second mutant GATA3 peptide sequence from (e); (e) the first mutant GATA3 peptide sequence from (b) and the second mutant GATA3 peptide sequence from (c); (f) the first mutant GATA3 peptide sequence from (b) and the second mutant GATA3 peptide sequence from (d); (g) the first mutant GATA3 peptide sequence from (b) and the second mutant GATA3 peptide sequence from (e); (h) the first mutant GATA3 peptide sequence from (c) and the second mutant GATA3 peptide sequence from (d); (i) a first mutant GATA3 peptide sequence from (c) and a second mutant GATA3 peptide sequence from (e); or (j) The first mutant GATA3 peptide sequence from (d) and the second mutant GATA3 peptide sequence from (e). 18. The pharmaceutical composition according to item 16 or 17, comprising: (Item 19) 19. The pharmaceutical composition according to any one of items 1 to 18, wherein the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprise a peptide in Table 5 and / or Table 6. (Item 20) 21. The pharmaceutical composition according to any one of Items 1 to 19, wherein the first mutant GATA3 peptide sequence comprises a first neoepitope of a GATA3 protein, and the second mutant GATA3 peptide sequence comprises a second neoepitope of a mutant GATA protein, the first mutant GATA3 peptide sequence is different from the second mutant GATA3 peptide sequence, the first neoepitope comprises at least one mutant amino acid, and the second neoepitope comprises the same mutant amino acid. each of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprising at least 8 consecutive amino acids, [Xaa] F -[Xaa] N -[Xaa] C or [Xaa] N -[Xaa] C -[Xaa] F where: each Xaa is an amino acid, [Xaa] N and [Xaa] C each comprising an amino acid sequence encoded by a different portion of the GATA3 gene, [Xaa] F is any amino acid sequence, [Xaa] N is encoded in a non-wild-type reading frame of the GATA3 gene, [Xaa] C contains at least one mutant amino acid and is encoded in a non-wild-type reading frame of the GATA3 gene; N is an integer from 0 to 100, C is an integer from 1 to 100, F is an integer from 0 to 100, 21. The pharmaceutical composition according to any one of items 1 to 20, wherein the sum of N and M is at least 8. (Item 22) [Xaa] F and F is an integer of 1 to 100, 1 to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. (Item 23) 23. The pharmaceutical composition according to item 22, wherein F is 3, 4 or 5. (Item 24) 24. The pharmaceutical composition according to any one of items 1 to 23, wherein each of the mutant GATA3 peptide sequences is present at a concentration of at least 50 μg / mL to 400 μg / mL. (Item 25) 25. The pharmaceutical composition according to any one of items 1 to 24, wherein the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprise a sequence in Table 1 or Table 2. (Item 26) 26. The pharmaceutical composition according to any one of items 1 to 25, further comprising an immunomodulator or adjuvant. (Item 27) 27. The pharmaceutical composition of claim 26, wherein the adjuvant is polyICLC. (Item 28) 1. A pharmaceutical composition comprising one or more mutant GATA3 peptide sequences, A pharmaceutical composition, wherein the one or more mutant GATA3 peptide sequences comprise a sequence selected from the group consisting of ESKIMFATLQRSSL, KPKRDGYMFLKAESKI, SMLTGPPARVPAVPFDLH, EPCSMLTGPPARVPAVPFDLH, LHFCRSSIMKPKRDGYMFLKAESKI, GPPARVPAVPFDLHFCRSSIMKPKRD, and KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH. (Item 29) 29. The pharmaceutical composition according to any one of items 1 to 28, comprising a pH adjusting agent present in a concentration of 0.1 mM to 1 mM. (Item 30) 29. The pharmaceutical composition according to any one of items 1 to 28, comprising a pH adjusting agent present in a concentration between 1 mM and 10 mM. (Item 31) 1. A method for synthesizing a GATA3 peptide, comprising: the peptide comprises a sequence of at least two consecutive amino acids selected from the group consisting of Xaa-Cys, Xaa-Ser, and Xaa-Thr, where Xaa is any amino acid; (a) coupling at least one dipeptide or derivative thereof with an amino acid or derivative thereof of a GATA3 peptide or derivative thereof to obtain a pseudoproline containing GATA3 peptide or derivative thereof, wherein the dipeptide or derivative thereof comprises a pseudoproline moiety; (b) coupling one or more selected amino acids, small peptides or derivatives thereof to said pseudoproline containing GATA3 peptide or derivative thereof; and (c) cleaving the pseudoproline containing GATA3 peptide or derivative thereof from the resin. (Item 32) 32. The method of claim 31, further comprising deprotecting the pseudoproline containing GATA3 peptide or a derivative thereof. (Item 33) 33. The method according to any one of items 31 to 32, wherein the amino acid or derivative thereof to which at least one dipeptide or derivative thereof is coupled is selected from the group consisting of Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, Tyr, His, and Val. (Item 34) The one or more selected amino acids, small peptides or derivatives thereof optionally coupled to the pseudoproline containing GATA3 peptide or derivative thereof may be selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc) ... 34. The method according to any one of items 31 to 33, comprising Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH. (Item 35) The N-terminal amino acid or a derivative thereof of the GATA3 peptide or a derivative thereof is selected from the group consisting of Fmoc-Ala-OH·HO, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OMpe)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc- 35. The method according to any one of items 31 to 34, wherein the nucleotide sequence is selected from the group consisting of Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH and Fmoc-His(Boc)-OH. (Item 36) The pseudoproline moiety is (a) Fmoc-Ser(tBu)-Ser(psi(Me, Me)pro)-OH, (b) Fmoc-Ala-Thr(psi(Me,Me)pro)-OH, (c)Fmoc-Glu(OtBu)-Ser(psi(Me, Me)pro)-OH, (d) Fmoc-Leu-Thr(psi(Me,Me)pro)-OH, (e)Fmoc-Leu-Cys(psi(Dmp,H)pro)-OH 36. The method according to any one of Items 31 to 35, wherein (Item 37) (a) Xaa-Ser is Ser-Ser, (b) Xaa-Ser is Glu-Ser; (c) Xaa-Thr is Ala-Thr; (d) Xaa-Thr is Leu-Thr, or (e) Xaa-Cys is Leu-Cys; 37. The method according to any one of Items 31 to 36. (Item 38) 31. A method for treating a subject having cancer, comprising administering to the subject the pharmaceutical composition according to any one of items 1 to 30. (Item 39) 1. A method for identifying a subject having cancer as a candidate for a therapeutic agent, comprising: identifying the subject as expressing a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele, and / or an HLA-B08:01 allele; The therapeutic agent is (a) at least one polypeptide comprising one or more mutant GATA3 peptide sequences, each of which comprises at least one mutant amino acid and is a fragment of at least 8 consecutive amino acids of a mutant GATA3 protein resulting from a mutation in the GATA3 gene of a cancer cell; or (b) at least one polynucleotide comprising a sequence encoding said at least one polypeptide; A method wherein each of the one or more mutant GATA3 peptide sequences or a portion thereof binds to a protein encoded by an HLA-A02:01 allele, an HLA-A24:02 allele, an HLA-A03:01 allele, an HLA-B07:02 allele and / or an HLA-B08:01 allele. (Item 40) 40. The method of claim 39, further comprising administering the therapeutic agent to the subject. (Item 41) 1. A method of treating a subject having cancer, comprising administering to the subject: (a) at least one polypeptide comprising a first mutant GATA3 peptide sequence and a second mutant GATA3 peptide sequence, (i) each of the first mutant GATA3 peptide sequence and the second mutant GATA3 peptide sequence comprises at least 8 consecutive amino acids of SEQ ID NO: 1; and (ii) the at least one polypeptide, wherein the C-terminal sequence of the first mutant GATA3 peptide sequence overlaps with the N-terminal sequence of the second mutant GATA3 peptide sequence, and wherein at least eight consecutive amino acids of SEQ ID NO: 1 comprise at least one amino acid of the sequence PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); or (b) administering a pharmaceutical composition comprising at least one polynucleotide comprising a sequence encoding said at least one polypeptide; The method wherein the HLA alleles expressed by the subject are unknown at the time of administration. (Item 42) At least 8 consecutive amino acids of SEQ ID NO: 1 have the sequence: Item 42. The method of item 41, comprising at least one amino acid of PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2). (Item 43) 43. The method of any one of items 41 to 42, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL). (Item 44) 44. The method of any one of items 41 to 43, wherein the subject has breast cancer that is resistant to anti-estrogen therapy, MSI breast cancer, metastatic breast cancer, Her2-negative breast cancer, Her2-positive breast cancer, ER-negative breast cancer, ER-positive breast cancer, PR-positive breast cancer, PR-negative breast cancer, or any combination thereof. (Item 45) 45. The method of item 44, wherein the breast cancer expresses a mutated estrogen receptor. (Item 46) 46. ​​The method of any one of items 41 to 45, further comprising administering at least one additional therapeutic agent or therapy. (Item 47) 47. The method of item 46, wherein the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. (Item 48) 48. The method of item 47, wherein the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, an anti-CD40 agent, letrozole, fulvestrant, a PI3 kinase inhibitor, and / or a CDK4 / 6 inhibitor. (Item 49) 48. The method of item 47, wherein the at least one additional therapeutic agent is palbociclib, ribociclib, abemaciclib, seliciclib, dinaciclib, milciclib, roniciclib, atubeciclib, bliciclib, ribiciclib, seliciclib, trilaciclib, voruciclib, or any combination thereof. (Item 50) and / or the at least one additional therapeutic agent is palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; arciliaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-aminothioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (lyubidin); flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atubeciclib; bliciclib; ribiciclib; trilaciclib (G1T28); or any combination thereof. (Item 51) The at least one additional therapeutic agent is selected from the group consisting of wortmannin, demethoxyviridin, LY294002, hibiscon C, idelalisib, copanlisib, duvelisib, taselisib, perifosine, buparlisib, duvelisib, alpelisib (BYL719), umbralisib, (TGR1202), copanlisib (BAY80-6946), PX-866, dactolisib, CUDC-907, voxtalisib (SAR245409, X L765), CUDC-907, ME-401, IPI-549, SF1126, RP6530, INK1117, pictilisib (GDC-0941), XL147 (SAR245408), Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477, or AEZS-136. (Item 52) 52. The method of any one of items 41 to 51, wherein the cancer is recurrent or metastatic breast cancer. (Item 53) 53. The method of any one of items 41 to 52, wherein the subject has experienced disease progression after endocrine therapy in combination with a CDK4 / 6 inhibitor or the subject has not previously received systemic therapy. (Item 54) 54. The method of any one of items 41 to 53, comprising determining the mutation status of the estrogen receptor gene in the cells of the subject. (Item 55) 55. The method of claim 54, wherein the cell is an isolated cell or a cell with increased expression of an estrogen receptor. [Brief explanation of the drawings]

[0177] [Figure 1] FIG. 1 shows an exemplary workflow for the determination of GATA3 epitopes capable of inducing CD8+ and / or CD4+ T cells.

[0178] [Figure 2] Figure 2 shows an exemplary experimental workflow for determining whether an epitope is processed and presented (top) and whether the epitope is recognized by T cells (bottom). This workflow confirmed that the GATA3 neoantigen was processed and presented (detected by mass spectrometry) and that the GATA3 neoantigen bound to HLA multimers could be recognized by recombinant T cell receptors (TCRs) expressed in Jurkat cell lines.

[0179] [Figure 3] Figure 3 shows an exemplary schematic of the workflow for detecting GATA3 neo-ORF epitopes by mass spectrometry. For peptide isolation, batch lysis was performed. For immunoprecipitation, an HLA class I pan-antibody (W6 / 32) was used.

[0180] [Figure 4] FIG. 4 shows an exemplary schematic diagram of the workflow for GATA3 antigen-specific expansion of CD8+ T cells.

[0181] [Figure 5] FIG. 5 shows a summary of experiments demonstrating that predicted GATA3 epitopes for HLA-A02 (left), HLA-B07 (center), and HLA-B08 (right) can be detected by mass spectrometry.

[0182] [Figure 6] Figure 6 is an illustration of the GATA3 neo ORF. The shaded regions represent portions of the GATA3 neo ORF sequence shared by all patients (common region) and some patients (variable region).

[0183] [Figure 7-1] FIG. 7A is an illustration of the GATA3 neo ORF sequence (SEQ ID NO: 2) with the variable region sequence (SEQ ID NO: 3) and the common region sequence (SEQ ID NO: 4).

[0184] [Figure 7-2] Figure 7B is a schematic diagram showing the GATA3 sequence (SEQ ID NO: 1) with the neo-ORF sequence (SEQ ID NO: 2), showing that three predicted HLA-02:01 epitopes, two predicted HLA-B07:02 epitopes, and one predicted HLA-B08:01 epitope were observed by mass spectrometry. This data indicates that the epitopes are targetable.

[0185] [Figure 7-3] FIG. 7C is an illustration of an example peptide design scheme for overlapping peptides (OLPs) spanning the entire GATA3 neo-ORF region.

[0186] [Figure 7-4] FIG. 7D is an exemplary amino acid sequence of the variable region of the GATA3 neo ORF (SEQ ID NO: 3).

[0187] [Figure 7-5] FIG. 7E is an exemplary amino acid sequence of the consensus region of the GATA3 neo ORF (SEQ ID NO: 4).

[0188] [Figure 8] Figure 8 is a graph showing the number of therapeutic Class I GATA3 neo-ORF epitopes versus the percentage of patients containing these epitopes. Most patients have 4-5 epitopes.

[0189] [Figure 9-1] FIG. 9A shows example results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples derived from human donors.

[0190] [Figure 9-2] Figure 9B shows example results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples from human donors.

[0191] [Figure 9-3] FIG. 9C shows example results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples derived from human donors.

[0192] [Figure 10-1] FIG. 10A shows example results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples derived from human donors.

[0193] [Figure 10-2] FIG. 10B shows example results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples from human donors.

[0194] [Figure 11] FIG. 11 shows FACS analysis of antigen-specific induction of IFNγ and TNFα levels in CD4+ cells derived from healthy HLA-A02:01 donors stimulated with APCs loaded with or without GATA3 neo-ORF peptides.

[0195] [Figure 12-1] FIG. 12A shows that the indicated peptides were soluble in pharmaceutical compositions containing 5 mM or 0.25 mM succinate, no DMSO, 5% dextrose in water (5DW), and no polyICLC at the indicated peptide concentrations.

[0196] [Figure 12-2] FIG. 12B shows that the indicated peptides were soluble in pharmaceutical compositions of 5 mM or 0.25 mM succinate, no DMSO, 5% dextrose in water (5DW), and poly-ICLC at the indicated peptide concentrations.

[0197] [Figure 12-3] Figure 12C shows that the indicated peptides were soluble in pharmaceutical compositions of 5 mM or 0.25 mM succinate at the indicated peptide concentrations, no DMSO, 5% dextrose in water (5DW), and polyICLC at the indicated peptide:polyICLC ratios.

[0198] [Figure 13] FIG. 13 shows the amino acid sequence of the consensus region of GATA3 frameshift mutations (SEQ ID NO: 4).

[0199] [Figure 14] FIG. 14 shows Kaplan-Meier survival curves for patients in the MSK-IMPACT breast cancer dataset.

[0200] [Figure 15] FIG. 15 shows the simulated number of presented epitopes per patient.

[0201] [Figure 16] FIG. 16 shows an alignment of the GATA3 wild-type and mutant nucleotide sequences.

[0202] [Figure 17] FIG. 17 shows an alignment of GATA3 wild-type and mutant amino acid sequences.

[0203] [Figure 18] FIG. 18 shows the plasmid map encoding the GATA3 mutation.

[0204] [Figure 19] FIG. 19 shows the multi-alignment of GATA3 mutant genes and DNA sequencing data of GATA3 mutant plasmid constructs.

[0205] [Figure 20] FIG. 20 shows the restriction enzyme digestion of the GATA3 mutant plasmid with AflII.

[0206] [Figure 21] FIG. 21 shows the expression of MHC class I and MHC class II in GATA3-transduced HEK293T cells.

[0207] 22A-22D show the expression profiles of HLA-A02 and MHC-ABC in GATA3 HEK293T cells transfected with HLA-A02.01, HLA-B07.02, and HLA-B08.01.

[0208] [Figure 22-1] FIG. 22A shows untransfected GATA3 HEK293T cells.

[0209] [Figure 22-2] FIG. 22B shows GATA3 HEK293T cells transfected with HLA-A02.01.

[0210] [Figure 22-3] FIG. 22C shows GATA3 HEK293T cells transfected with HLA-B07.02.

[0211] [Figure 22-4]Figure 22D shows GATA3 HEK293T cells transfected with HLA-B08.01.

[0212] [Figure 23] Figure 23 shows the detection of predicted peptide epitopes derived from the common region of GATA3 neo ORF stably expressed in HEK293T cells. The light gray and black sequences represent the variable and common regions of GATA3 neo ORF, respectively.

[0213] [Figure 24-1] FIG. 24A shows the MS / MS spectra of the endogenously processed peptide epitope SMLTGPPARV (bottom) and its corresponding synthetic peptide (top).

[0214] [Figure 24-2] FIG. 24B shows a comprehensive plot of the MS / MS spectral matches.

[0215] [Figure 25-1] FIG. 25A shows the MS / MS spectra of the endogenously processed peptide epitope MLTGPPARV (bottom) and its corresponding synthetic peptide (top).

[0216] [Figure 25-2] FIG. 25B shows a comprehensive plot of the spectral matches.

[0217] [Figure 26-1] FIG. 26A shows the MS / MS spectra of the endogenously processed peptide epitope KPKRDGYMF (bottom) and its corresponding synthetic peptide (top).

[0218] [Figure 26-2] Figure 26B shows a comprehensive plot of the spectral matches.

[0219] [Figure 27-1]FIG. 27A shows the MS / MS spectra of the endogenously processed peptide epitope KPKRDGYMFL (bottom) and its corresponding synthetic peptide (top).

[0220] [Figure 27-2] Figure 27B shows a comprehensive plot of the spectral matches.

[0221] [Figure 28-1] FIG. 28A shows the MS / MS spectra of the endogenously processed peptide epitope ESKImFATL (bottom) and its corresponding synthetic peptide (top).

[0222] [Figure 28-2] Figure 28B shows a comprehensive plot of the spectral matches.

[0223] [Figure 29-1] FIG. 29A shows representative induction of CD8+ responses by GATA3 neo-ORF-specific peptides (FLT-mDC GATA3 Stim2 multimers).

[0224] [Figure 29-2] FIG. 29B shows a negative control with no induction of CD8+ responses in PBMCs and dendritic cells.

[0225] [Figure 30-1] FIG. 30A shows the induction of antigen-specific CD4 T cells without peptide.

[0226] [Figure 30-2] FIG. 30B shows the induction of antigen-specific CD4 T cells using GATA3 neo-ORF-specific peptides.

[0227] 31A-31D show GATA3-specific CD8+ T cells by multimer staining.

[0228] [Figure 31-1]FIG. 31A shows that after long-term stimulation of healthy donor HD47, an average of 1.16% positive GATA3-specific CD8+ T cells were observed.

[0229] [Figure 31-2] FIG. 31B shows that after long-term stimulation of healthy donor HD50, an average of 1.29% positive GATA3-specific CD8+ T cells were observed.

[0230] [Figure 31-3] FIG. 31C shows that after long-term stimulation of healthy donor HD51, an average of 1.9% positive GATA3-specific CD8+ T cells were observed.

[0231] [Figure 31-4] FIG. 31D shows that after long-term stimulation of healthy donor HD51 with different concentrations of peptides than in FIG. 31C, an average of 4.5% positive GATA3-specific CD8+ T cells were observed.

[0232] [Figure 32] Figure 32 shows a comparison of the caspase-3-positive percentage of live target cells. Four different GATA3-induced healthy donor PBMCs (1-4) were co-cultured with HEK293T cells transduced with a GATA3 mutation (GATA3Trd) or untransduced HEK293T cells (NoTRd293T) as a negative control.

[0233] [Figure 33] FIG. 33 shows a significant difference between GATA3-transduced and non-transduced HEK293T cells.

[0234] [Figure 34] FIG. 34 shows the difference in CD107a expression in CD8+ T cells co-cultured with GATA3-transduced or non-transduced HEK293T cells.

[0235] [Figure 35]Figure 35 shows the difference in IFN-γ concentration between GATA3-transduced and non-transduced HEK293T cells under co-culture conditions with GATA3-induced T cells.

[0236] [Figure 36] Figure 36 shows an overview of GATA3-specific TCR cloning, as described in detail in Example 26.

[0237] [Figure 37] Figure 37 shows an exemplary method for generating GATA3-specific TCR-transduced Jurkats and PBMCs, as described in detail in Example 26.

[0238] [Figure 38] FIG. 38 shows an outline of a functional assay using TCR-transduced Jurkats.

[0239] [Figure 39] FIG. 39 shows GATA3-specific CD8+ T cells by multimer staining for sorting.

[0240] [Figure 40] FIG. 40 shows a GATA3-specific TCR construct for lentivirus.

[0241] [Figure 41-1] FIG. 41A shows a multi-alignment of the GATA3 TCR alpha sequence and the wild-type DNA sequence.

[0242] [Figure 41-2] FIG. 41B shows a multi-alignment of the GATA3 TCR beta sequence and the wild-type DNA sequence.

[0243] [Figure 42] Figure 42 shows restriction enzyme digestion of the GATA3 TCR plasmid with AflII.

[0244] [Figure 43] FIG. 43 shows GATA3-specific TCR-transduced Jurkats stained with GATA3 multimer PE and GATA3 multimer BV650.

[0245] [Figure 44] FIG. 44 shows a GATA3-specific TCR peptide titration assay.

[0246] [Figure 45] FIG. 45 shows an IL-2 release assay of Jurkat cells transduced with a GATA3-specific TCR and target cells transduced with a GATA3 mutation.

[0247] [Figure 46] Figure 46 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 14 amino acids and the sequence ESKIMFATLQRSSL. The peptide has a molecular formula of C70H119N19O22S and a molecular weight of 1610.89 g / mol. The peptide is in the form of the trifluoroacetic acid (TFA) salt.

[0248] [Figure 47] Figure 47 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 16 amino acids and has the sequence KPKRDGYMFLKAESKI. The peptide has a molecular formula of C87H143N23O23S and a molecular weight of 1911.30 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0249] [Figure 48] Figure 48 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 18 amino acids and has the sequence SMLTGPPARVPAVPFDLH. The peptide has a molecular formula of C87H137N23O23S and a molecular weight of 1905.25 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0250] [Figure 49] Figure 49 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 21 amino acids and has the sequence EPCSMLTGPPARVPAVPFDLH. The peptide has a molecular formula of C100H156N26O28S2 and a molecular weight of 2234.62 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0251] [Figure 50] Figure 50 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 25 amino acids and has the sequence LHFCRSSIMKPKRDGYMFLKAESKI. The peptide has a molecular formula of C134H217N37O34S3 and a molecular weight of 2986.62 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0252] [Figure 51] Figure 51 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 26 amino acids and the sequence GPPARVPAVPFDLHFCRSSIMKPKRD. The peptide has a molecular formula of C131H209N39O33S2 and a molecular weight of 2922.47 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0253] [Figure 52] Figure 52 shows the stereochemistry of an exemplary GATA3 neo-ORF peptide. The peptide consists of 33 amino acids and the sequence KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH. The peptide has a molecular formula of C173H274N48O46S4 and a molecular weight of 3890.63 g / mol. The peptide is in the form of a trifluoroacetic acid (TFA) salt.

[0254] [Figure 53] Figure 53 shows BTK antigenic peptide-specific CD8+ T cell responses using PBMC samples from human donors.

[0255] [Figure 54] Figure 54 shows EGFR antigenic peptide-specific CD8+ T cell responses using PBMC samples derived from human donors. DETAILED DESCRIPTION OF THE INVENTION

[0256] GATA3 is a gene highly expressed in breast cancer and is one of the most frequently mutated genes in these cancers. The most common class of mutations in this gene are insertions and deletions between the nucleotides encoding amino acid 393 and amino acid 445 (the natural stop codon). When these mutations shift the open reading frame to the +1 frame, they lead to an extended novel reading frame ("neo-ORF") that introduces at least 61 and as many as 113 amino acids not normally expressed in healthy cells. While 61 amino acids are shared among all patients (conserved region), each patient possesses 0–52 additional amino acids (variable region). Thus, the epitope processed and presented from this neo-ORF is a neo-antigen shared among some or all patients carrying this same class of mutation. GATA3 neo-ORF appears to be an adverse prognostic factor in breast cancer. While GATA3 wild-type is a highly expressed gene, GATA3 neo-ORF retains high expression. The GATA3 neo-ORF is translated and associated with increased risk of breast cancer.

[0257] In some embodiments, long overlapping peptides (OLPs) spanning the entire neo-ORF can be used to treat cancer. In some aspects, the OLPs described herein are designed to contain epitopes at the termini of the peptide, which simplifies the processing and presentation process (since only one cleavage event is required). In some aspects, short peptides (e.g., 9-11 amino acids) that bind to MHC class I proteins can be administered to a subject to treat cancer. Using the techniques described herein, many neo-antigens can be targeted without the need to select patients based on their HLA composition.

[0258] In some embodiments, the peptides described herein can include modifications (e.g., lipidation) that can increase immunogenicity. In some embodiments, polynucleotides (e.g., polybodies) encoding polypeptides encoded by the entire GATA3 neo ORF are provided. In some embodiments, cell-based therapies, such as engineered T cells expressing TCRs that target specific epitopes, can be used to treat subjects with cancer.

[0259] Disclosed herein are synthetic long peptides (SLPs) spanning the consensus region of the GATA3 protein. These peptides are soluble in the formulations described herein and are compatible with poly-ICLC for sc injection. High purity and synthetic yield for one or more of these peptides can be achieved by employing pseudoproline blocks during solid-phase peptide synthesis (SPPS). Purification conditions for each of these peptides have also been developed.

[0260] Described herein are novel immunotherapeutic agents and their uses based on the discovery of neoantigens that arise from mutational events unique to an individual's tumor. Accordingly, the disclosure described herein provides peptides, polynucleotides encoding the peptides, and peptide-binding agents that can be used, for example, to stimulate an immune response against tumor-associated antigens or neoepitopes, to create immunogenic compositions or cancer vaccines for use in treating disease.

[0261] The following description and examples illustrate in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will appreciate that there are numerous variations and modifications of the present disclosure that fall within the scope of the present invention.

[0262] All terms are intended to be understood as they are understood by one of ordinary skill in the art. 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 to which this disclosure belongs.

[0263] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0264] While various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0265] The following definitions supplement those in the art and are intended to be inclusive of any related or unrelated case, for example, any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. definition

[0266] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly dictates otherwise.

[0267] In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrase "A, B and / or C" or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B and C." The term "or" may be used conjunctively or disjunctively, unless the context specifically dictates disjunctive use.

[0268] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of the measurement system, i.e., how the value is measured or determined. For example, "about" can mean within one standard deviation, or more than one standard deviation, according to practice in the art. Alternatively, "about" can mean within 20% or less, 10% or less, 5% or less, or 1% or less of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or more preferably within 2-fold of a value. When a particular value is described in this application or claims, unless otherwise stated, the term "about" should be construed to mean within an acceptable error range of the particular value.

[0269] As used in this application and in the claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or containing (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be practiced with respect to any method or composition of the disclosure, and conversely, any method or composition of the disclosure can be practiced with respect to any embodiment discussed herein. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0270] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0271] The "major histocompatibility complex" or "MHC" is a cluster of genes involved in controlling the cellular interactions that result in physiological immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3 rd Ed., Raven Press, New York (1993). "Major histocompatibility complex (MHC) protein or molecule," "MHC molecule," "MHC protein," or "HLA protein" is understood to mean a protein capable of binding to peptides resulting from proteolytic cleavage of a protein antigen, presenting potential lymphocyte epitopes (e.g., T-cell epitopes and B-cell epitopes), and transporting them to the cell surface, where they can be presented to specific cells, particularly cytotoxic T lymphocytes, helper T cells, or B cells. The major histocompatibility complex within the genome contains gene regions whose expressed gene products on the cell surface are important for binding to and presenting endogenous and / or foreign antigens and thus regulating immunological processes. The major histocompatibility complex is divided into two groups of genes encoding different proteins: MHC class I molecules and MHC class II molecules. The cellular biology and expression patterns of the two MHC classes are adapted to their distinct roles.

[0272] "Human leukocyte antigens" or "HLA" are human class I or class II major histocompatibility complex (MHC) proteins (see, e.g., Stites, et al., Immunology, 8 th Ed., Lange Publishing, Los Altos, Calif. (1994).

[0273] As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to a polymer of amino acid residues, usually L-amino acids, connected one to the other by a peptide bond between the α-amino and carboxyl groups of adjacent amino acids. Polypeptides and peptides include, but are not limited to, "mutant peptides," "neo-antigenic peptides," and "neo-antigenic peptides." Polypeptides or peptides can be of various lengths, in either their neutral (uncharged) or salt form, and can be free of or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein. A "mature protein" is a protein that is full-length and, optionally, contains glycosylation or other modifications typical of proteins in a given cellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can contain synthetic amino acids in place of one or more naturally occurring amino acids.Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycerin, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, isopropyl alcohol, methylalanine ... Examples of amino acids that may be used include lysine-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The present disclosure further contemplates that expression of the polypeptides described herein in engineered cells may be accompanied by post-translational modification of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0274] A peptide or polypeptide can include at least one flanking sequence. As used herein, the term "flanking sequence" refers to a fragment or region of a peptide that is not part of the epitope.

[0275] An "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" is a peptide that binds to an HLA molecule and elicits a cell-mediated or humoral response, such as the induction of cytotoxic T lymphocytes (CTLs, e.g., CD8 + )), helper T lymphocytes (Th (e.g., CD4 + )) and / or B lymphocyte responses. Thus, the immunogenic peptides described herein are capable of binding to the appropriate HLA molecule and subsequently inducing a CTL (cytotoxic) response or an HTL (and humoral) response against the peptide.

[0276] "Neoantigen" refers to a class of tumor antigens that arise from tumor-specific alterations of proteins. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, protein sequence substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides.

[0277] The term "residue" refers to an amino acid residue or amino acid mimetic residue that is incorporated into a peptide or protein by an amide bond or amide bond mimetic, or into a nucleic acid (DNA or RNA) that encodes the amino acid or amino acid mimetic.

[0278] A "neoepitope," "tumor-specific neoepitope," or "tumor antigen" refers to an epitope or antigenic determinant region that is absent from reference non-diseased cells, e.g., non-cancerous cells or germline cells, but is found in diseased cells, e.g., cancer cells. This includes situations where the corresponding epitope is found in normal non-diseased cells or germline cells, but one or more mutations in diseased cells, e.g., cancer cells, result in a change in the sequence of the epitope, creating a neoepitope. As used herein, the term "neoepitope" refers to an antigenic determinant region within a peptide or neoantigenic peptide. A neoepitope may include at least one "anchor residue" and at least one "anchor residue-adjacent region." A neoepitope may further include a "separation region." The term "anchor residue" refers to an amino acid residue that binds to a specific pocket on HLA, conferring specificity to the interaction with HLA. In some cases, the anchor residue may be located at a canonical anchor position. In other cases, anchor residues may reside at non-canonical anchor positions. Neoepitopes can bind to HLA molecules through primary and secondary anchor residues that protrude into pockets in the peptide-binding groove. Within the peptide-binding groove, specific amino acids form pockets that correspond to the corresponding side chains of the anchor residues of the presented neoepitope. Peptide-binding preferences exist between different alleles of both HLA I and HLA II molecules. HLA class I molecules bind short neoepitopes, whose N- and C-termini are tethered to pockets located at the ends of the neoepitope-binding groove. While the majority of HLA class I-binding neoepitopes are approximately 9 amino acids long, longer neoepitopes can be tailored by extending their central portions, resulting in binding neoepitopes of approximately 8–12 amino acids. Neoepitopes that bind to HLA class II proteins are not constrained in size and can vary from approximately 16 to 25 amino acids. The neoepitope binding groove in HLA class II molecules is open at both ends, which allows the binding of peptides with relatively longer lengths.Although the core 9-amino acid residue long segment contributes most to neoepitope recognition, the anchor residue-flanking region is also important for peptide specificity for HLA class II alleles. In some cases, the anchor residue-flanking region is the N-terminal residue. In other cases, the anchor residue-flanking region is the C-terminal residue. In yet other cases, the anchor residue-flanking region is both the N-terminal and C-terminal residues. In some cases, the anchor residue-flanking region is flanked by at least two anchor residues. An anchor residue-flanking region that is flanked by anchor residues is a "separation region."

[0279] " Reference " can be used to correlate and compare the results obtained by the method of the present disclosure from tumor specimen. Typically, " reference " can be obtained based on one or more normal specimens, particularly specimens that are not affected by cancer disease, taken from either a patient or one or more individuals, for example, healthy individuals, particularly individuals of the same species. " Reference " can be determined experimentally by testing a sufficient number of normal specimens.

[0280] An "epitope" is a group of molecular features, such as primary, secondary, and tertiary peptide structure and charge, that together form a recognition site, e.g., a site recognized by an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor. Alternatively, an epitope can be defined as a series of amino acid residues involved in recognition by a particular immunoglobulin or in the context of a T cell, and that are required for recognition by a T cell receptor protein, a chimeric antigen receptor, and / or a major histocompatibility complex (MHC) receptor. A "T cell epitope" is understood to mean a peptide sequence that can be bound by a class I or II MHC molecule in the form of a peptide-presenting MHC molecule or MHC complex, and then, in this form, recognized and bound by T cells, such as T lymphocytes or helper T cells. Epitopes can be prepared by isolation from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes can include artificial amino acid residues, "amino acid mimetics," e.g., D-isomers of naturally occurring L-amino acid residues, or non-naturally occurring amino acid residues, e.g., cyclohexylalanine. Throughout this disclosure, epitopes are sometimes referred to as peptides or peptide epitopes. It should be understood that proteins or peptides containing the epitopes or analogs described herein, as well as additional amino acids, are also within the scope of this disclosure. In certain embodiments, peptides comprise fragments of antigens. In certain embodiments, the peptides of the present disclosure are length-restricted. Length-restricted embodiments occur when a protein or peptide containing an epitope described herein contains a region (i.e., a contiguous stretch of amino acid residues) that shares 100% identity with a native sequence. To avoid defining an epitope from a read-through, e.g., for the entire native molecule, there is a length restriction on any region that shares 100% identity with a native peptide sequence.Thus, for peptides comprising an epitope described herein and a region having 100% identity to a native peptide sequence, the region having 100% identity to the native sequence will generally have a length of less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an "epitope" as described herein is comprised of a peptide having a region having fewer than 51 amino acid residues, e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue, that has 100% identity to a native peptide sequence in any increment down to 5 amino acid residues.

[0281] The nomenclature used to describe peptides or proteins follows the conventional convention of presenting the amino group at the left (amino- or N-terminus) and the carboxyl group at the right (carboxyl- or C-terminus) of each amino acid residue. When referring to amino acid residue positions within a peptide epitope, they are numbered from amino to carboxyl, with the residue located at the amino terminus of the epitope, or of the peptide or protein of which it may be a part, being number 1. In formulas representing selected specific embodiments of the present disclosure, the amino and carboxyl terminal groups are not specifically shown, but are in the form they would be in at physiological pH unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or single-letter designation. The L-form of an amino acid residue is represented by a single capital letter or by the first letter of a three-letter designation with a capital letter, and the D-form for amino acid residues having a D-form is represented by a single lowercase letter or by a lowercase three-letter designation. However, when three-letter designations or full names are used without capital letters, they may also refer to L-amino acid residues. Glycine is referred to simply as "Gly" or "G" because it has no asymmetric carbon atom. The amino acid sequences of peptides presented herein are generally represented using standard single-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0282] The term "mutation" refers to a change or difference (nucleotide substitution, addition, or deletion) in a nucleic acid sequence compared to a reference. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and therefore are not passed on to offspring. These changes can (but do not always) cause cancer or other diseases. In some embodiments, the mutation is a nonsynonymous mutation. The term "nonsynonymous mutation" refers to a mutation that results in an amino acid change, such as an amino acid substitution, in the translation product, e.g., a nucleotide substitution. A "frameshift" occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. Different mutations of a gene can acquire the same altered reading frame.

[0283] A "conservative" amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of tyrosine with phenylalanine is a conservative substitution.Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate peptide function are well known in the art.

[0284] As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, e.g., an HLA-binding peptide and a class I or II HLA. Dis the dissociation constant and has units of molar concentration. The affinity constant is the reciprocal of the dissociation constant. Affinity constant is sometimes used as a general term to describe the chemical entity. It is a direct measure of binding energy. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. Affinity is measured using the inhibitory concentration 50 (IC), which is the concentration at which 50% of the peptide is displaced. 50 ) Similarly, ln(IC 50 ) is IC 50 It refers to the natural logarithm of K. off refers to the dissociation rate constant, for example, for the dissociation of an HLA-bound peptide with class I or II HLA. Throughout this disclosure, the results of "binding data" are referred to as "IC 50 " IC 50 is the concentration of test peptide in the binding assay at which 50% inhibition of binding of the labeled reference peptide is observed. Given the conditions under which the assay is performed (i.e., limiting HLA protein and labeled reference peptide concentrations), these values ​​are D Assays for determining binding are well known in the art and are described, for example, in PCT Publications WO94 / 20127 and WO94 / 03205, as well as other publications, for example, Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding may be expressed relative to binding by a reference standard peptide. For example, the IC of the reference standard peptide 50 For that IC 50Binding may also be determined using other assay systems, including those that use live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), those using cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), those using immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), those using ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), and those using surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); those using high-flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)); and those using measurements of class I MHC stabilization or association (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)). "Cross-reactive binding" indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

[0285] The term "derived" and its grammatical equivalents when used to discuss epitopes are synonymous with "prepared" and its grammatical equivalents. Derived epitopes can be isolated from natural sources, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can contain artificial amino acid residues, "amino acid mimetics," e.g., D-isomers of naturally occurring L-amino acid residues, or non-natural amino acid residues, e.g., cyclohexylalanine. Derived or prepared epitopes can also be analogs of native epitopes.

[0286] "Native" or "wild-type" sequence refers to a sequence found in nature. Such a sequence may naturally include a longer sequence.

[0287] "Receptor" is understood to mean a biological molecule or molecular configuration that can bind to a ligand. Receptors can function to transmit information about cells, cell formations, or organisms. A receptor comprises at least one receptor unit, where each receptor unit can be, for example, a protein molecule. A receptor has a structure complementary to that of a ligand and can complex with the ligand as a binding partner. The information is transmitted, in particular, by a conformational change of the receptor after complexation of the ligand on the cell surface. In some embodiments, receptors are understood to mean, in particular, proteins of MHC class I and II that can form receptor / ligand complexes with ligands, in particular peptides or peptide fragments of a suitable length.

[0288] A "ligand" is understood to mean a molecule having a structure complementary to that of a receptor and capable of forming a complex with the receptor. In some embodiments, a ligand is understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence such that the peptide or peptide fragment can form a complex with an MHC class I or MHC class II protein.

[0289] In some embodiments, "receptor / ligand complex" is also understood to mean a "receptor / peptide complex" or "receptor / peptide fragment complex" comprising a class I or class II peptide-presenting or peptide fragment-presenting MHC molecule.

[0290] "Synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., a man-made peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion proteins."

[0291] The term "motif" refers to a pattern of residues in an amino acid sequence of a defined length, e.g., a peptide less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, e.g., about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for class I HLA motifs and about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for class II HLA motifs, that is recognized by a particular HLA molecule. Motifs typically differ for each HLA protein encoded by a given human HLA allele. These motifs differ in their patterns of primary and secondary anchor residues. In some embodiments, MHC class I motifs distinguish peptides 9, 10, or 11 amino acid residues in length.

[0292] As used herein, the term "naturally occurring" and its grammatical equivalents refer to an entity that can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and that can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory is naturally occurring.

[0293] According to the present disclosure, the term "vaccine" relates to a pharmaceutical preparation (composition) or product that, upon administration, induces an immune response, e.g., a cellular or humoral immune response, that recognizes and attacks pathogens or diseased cells, e.g., cancer cells. Vaccines can be used for the prevention or treatment of disease. The term "individualized cancer vaccine" or "personalized cancer vaccine" refers to a specific cancer patient, meaning that the cancer vaccine is adapted to the needs or special circumstances of the individual cancer patient.

[0294] "Antigen processing" or "processing" and grammatical equivalents refer to the degradation of a polypeptide or antigen into processing products that are fragments of said polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association of one or more of these fragments with an MHC molecule (e.g., via binding) for presentation to specific T cells by a cell, e.g., an antigen-presenting cell.

[0295] Antigen-presenting cells (APCs) are cells that present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCs can activate antigen-specific T cells. Professional antigen-presenting cells are highly efficient at internalizing antigens by either phagocytosis or receptor-mediated endocytosis and then presenting the antigen fragments bound to class II MHC molecules on their membrane. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. As a result, additional costimulatory signals are produced by the antigen-presenting cell, leading to T cell activation. The expression of costimulatory molecules is a defining characteristic of professional antigen-presenting cells. The main types of professional antigen-presenting cells are dendritic cells (which have the broadest antigen-presenting range and are perhaps the most important antigen-presenting cells), macrophages, B cells, and certain activated epithelial cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both the MHC class II and MHC class I antigen-presenting pathways. It is well known that dendritic cells are potent inducers of immune responses and that activation of these cells is an essential step in the induction of antitumor immunity. Dendritic cells are conveniently broadly classified as "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with high antigen uptake and processing capacity, which correlates with high expression of Fc receptors (FcRs) and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers, but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0296] As used herein, the term "identical" and its grammatical equivalents, or "sequence identity" in the context of two nucleic acid or amino acid sequences of a polypeptide, refers to residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions, over which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison is described in Smith and Waterman, Adv. Appl. Math., 2:482. (1981) local homology algorithm; by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA, 85:2444 (1988); by computer implementations of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program by Intelligentics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA), the CLUSTAL program being used by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989);Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988);Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference polypeptide or fragment thereof as measured, for example, by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described relative to a starting nucleic acid, e.g., they may have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% sequence identity to a reference nucleic acid or fragment thereof, as determined, for example, by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, that percentage of residues in the smaller molecule will meet matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0297] The term "substantially identical" and its grammatical equivalents, when applied to a nucleic acid sequence or an amino acid sequence, means that the nucleic acid or amino acid sequence contains a sequence having at least 90% or more sequence identity, or at least 95%, at least 98%, and at least 99% sequence identity compared to a reference sequence using standard parameters with the above-mentioned programs, such as BLAST. For example, the BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where portions of the polynucleotide sequences within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage sequence identity. In embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues in length, over a region of at least about 100 residues, and in some embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding region.

[0298] As used herein, the term " vector " refers to the construct that can deliver and usually express one or more genes or sequences of interest in host cell.Examples of vector include but are not limited to virus vector, naked DNA or RNA expression vector, plasmid, cosmid or phage vector, DNA or RNA expression vector with cationic condensing agent, and DNA or RNA expression vector that is encapsulated in liposome.

[0299] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. In some embodiments, an "isolated polynucleotide" encompasses a PCR or quantitative PCR reaction, including a polynucleotide amplified in a PCR or quantitative PCR reaction.

[0300] The terms "isolated," "biologically pure," or their grammatical equivalents refer to a substance that is substantially or essentially free from components that normally accompany the substance when found in its native state. Thus, the isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their native environment. An "isolated" epitope refers to an epitope that does not contain the entire sequence of the antigen from which the epitope is derived. Typically, an "isolated" epitope is free of additional amino acid residues that result in a sequence that is 100% identical over the entire length of the native sequence. The native sequence may be the sequence of a tumor-associated antigen from which the epitope is derived. Thus, the term "isolated" means that a substance has been removed from its original environment (e.g., the natural environment if the substance is naturally occurring). An "isolated" nucleic acid is a nucleic acid that has been removed from its natural environment. For example, a polynucleotide or peptide that naturally occurs in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector, and / or such polynucleotides or peptides may be part of a composition, but may still be "isolated" in that such vectors or compositions are not part of their natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically.

[0301] As used herein, the term "substantially purified" and grammatical equivalents refer to a nucleic acid sequence, polypeptide, protein or other compound that is essentially free, i.e., more than about 50% free, more than about 70% free, more than about 90% free, of polynucleotides, proteins, polypeptides and other molecules with which the nucleic acid, polypeptide, protein or other compound is naturally associated.

[0302] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0303] The terms "polynucleotide," "nucleotide," "nucleic acid," "polynucleic acid," or "oligonucleotide," and their grammatical equivalents, are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Thus, these terms include double- and single-stranded DNA, triplex DNA, and double- and single-stranded RNA. It also includes modified, e.g., by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also intended to include molecules containing non-naturally occurring or synthetic nucleotides as well as nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into cells, e.g., by transfection, transformation, or transduction. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, polynucleotides and nucleic acids can be in vitro-transcribed mRNA. In some embodiments, the polynucleotide administered using the methods of the present disclosure is mRNA.

[0304] As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art, such as calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted biolistics (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al. al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after growing the infectious particles in suitable packaging cells, many of which are commercially available.

[0305] Nucleic acids and / or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" if their encoding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules are called homologs. For example, any naturally occurring protein described herein can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of identity between sequences that is useful for establishing homology varies depending on the nucleic acid and protein in question, but only about 25% sequence identity is typically used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or higher, can also be used to establish homology. Methods for determining percentage sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are publicly available.

[0306] The term "subject" refers to any animal (e.g., mammal), including but not limited to, humans, non-human primates, dogs, cats, rodents, etc., who will be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0307] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and thus can prevent the onset of a disease or disorder, can delay the onset of a disease or disorder, can slow the progression of a disease or disorder, can relieve to some extent one or more symptoms associated with a disease or disorder, can reduce morbidity or mortality, can improve quality of life, or a combination of such effects.

[0308] The terms "treating" or "treatment" or "to treat" or "alleviating" or "alleviating" refer to both (1) therapeutic measures that cure, delay, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those susceptible to the disorder; and those in whom the disease is to be prevented.

[0309] "Pharmaceutically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible.

[0310] "Pharmaceutical excipients" or "excipients" include substances such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, etc. A "pharmaceutical excipient" is a pharmaceutically acceptable excipient. Neoantigens and their uses

[0311] One of the most significant obstacles to the development of curative and tumor-specific immunotherapy is the identification and selection of highly specific, limited tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific antigen class. Neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to the technical challenges of identifying them, optimizing their selection, and producing them for use in vaccines or immunogenic compositions. These problems can be addressed by identifying neoplastic / tumor mutations that are present at the DNA level in the tumor but not in matched germline samples from a high percentage of subjects with cancer; analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate multiple neoantigenic T cell epitopes that are expressed in the neoplasm / tumor and bind to a high percentage of patient HLA alleles; and synthesizing multiple neoantigenic peptides selected from the set of all neoantigenic peptides and predicted binding peptides for use in cancer vaccines or immunogenic compositions suitable for treating a high percentage of subjects with cancer.

[0312] For example, translating peptide sequencing information into therapeutic vaccines may involve predicting mutant peptides capable of binding to HLA molecules for a high percentage of individuals. To efficiently select specific mutations for use as immunogens, it is necessary to be able to predict which mutant peptides will efficiently bind to a high percentage of a patient's HLA alleles. Recently, neural network-based learning techniques using validated binding and non-binding peptides have improved the accuracy of prediction algorithms for the major HLA-A and -B alleles. However, even with improved neural network-based algorithms to encode HLA-peptide binding rules, several factors limit the predictive power of peptides presented on HLA alleles.

[0313] Another example of translating peptide sequencing information into therapeutic vaccines is the formulation of drugs as long peptide multi-epitope vaccines. Targeting as many mutant epitopes as feasible exploits the immune system's enormous capabilities, prevents opportunities for immune evasion through downregulation of immune-targeted gene products, and compensates for the known accuracy of epitope prediction methods. Synthetic peptides provide a useful tool for efficiently preparing multiple immunogens and rapidly translating the identification of mutant epitopes into effective vaccines. Peptides can be easily chemically synthesized and purified using reagents free of contaminating bacterial or animal substances. Their small size allows for a clear focus on the mutated region of the protein and also reduces irrelevant antigen competition from other components (non-mutated proteins or viral vector antigens).

[0314] Another example of the translation of peptide sequencing information into therapeutic vaccines is its combination with a strong vaccine adjuvant. Effective vaccines may require a strong adjuvant to initiate an immune response. For example, poly-ICLC, an agonist for TLR3 and the RNA helicase domains of MDA5 and RIG3, has demonstrated several desirable properties for a vaccine adjuvant. These properties include inducing local and systemic activation of immune cells in vivo, producing stimulatory chemokines and cytokines, and stimulating antigen presentation by DCs. Furthermore, poly-ICLC has been shown to induce sustained CD4 activation in humans. + and CD8 + Importantly, striking similarities in upregulation of transcriptional and signaling pathways were observed in subjects vaccinated with poly-ICLC and in volunteers who received a highly effective, replicative yellow fever vaccine. Furthermore, >90% of ovarian cancer patients immunized with poly-ICLC in combination with the NYESO-1 peptide vaccine (in addition to Montanide) showed increased CD4 + and CD8 +Poly-ICLC has demonstrated induction of T cell and antibody responses to the peptide in a recent Phase 1 study. Additionally, poly-ICLC has been extensively tested in over 25 clinical trials to date and has demonstrated a relatively benign toxicity profile.

[0315] In some aspects, provided herein are compositions comprising a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein; compositions comprising a polynucleotide encoding the first peptide and the second peptide; compositions comprising one or more APCs comprising the first peptide and the second peptide; or compositions comprising a first T cell receptor (TCR) specific for a first neoepitope in a complex with an HLA protein and a second TCR specific for a second neoepitope in a complex with an HLA protein, wherein the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation.

[0316] In some aspects, provided herein are compositions comprising a first peptide comprising a first neoepitope of a region of a protein and a second peptide comprising a second neoepitope of the same region of the protein, wherein the first neoepitope and the second neoepitope comprise at least one amino acid of the same region; compositions comprising polynucleotides encoding the first peptide and the second peptide; compositions comprising one or more APCs comprising the first peptide and the second peptide; or compositions comprising a first T cell receptor (TCR) specific for a first neoepitope in a complex with an HLA protein and a second TCR specific for a second neoepitope in a complex with an HLA protein, wherein the first peptide is different from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation.

[0317] In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the first peptide and the second peptide are different molecules. In some embodiments, the first neoepitope comprises a first neoepitope in a region of the same protein, and the second neoepitope comprises a second neoepitope in a region of the same protein. In some embodiments, the first neoepitope and the second neoepitope comprise at least one amino acid in the same region. In some embodiments, a region of a protein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 consecutive amino acids of the protein. In some embodiments, the region of the protein comprises at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 consecutive amino acids of the protein. In some embodiments, the first neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex, and in some embodiments, the first neoepitope is a first neoepitope peptide processed from a first peptide, and / or the second neoepitope is a second neoepitope peptide processed from a second peptide.In some embodiments, the first neoepitope is shorter in length than the first peptide, and / or the second neoepitope is shorter in length than the second peptide. In some embodiments, the first neoepitope peptide is processed by an antigen-presenting cell (APC) containing the first peptide, and / or the second neoepitope peptide is processed by an APC containing the second peptide. In some embodiments, the first neoepitope is CD8. + In some embodiments, the second neoepitope activates CD4 T cells. + In some embodiments, the second neoepitope activates CD8 T cells. + In some embodiments, the first neoepitope activates CD4 T cells. + In some embodiments, CD4 + The TCR of the T cell binds to a class II HLA-peptide complex containing the first or second peptide. In some embodiments, the CD8 + The TCR of the T cell binds to a class I HLA-peptide complex containing the first or second peptide. In some embodiments, CD4 + The TCR of the T cell binds to a class I HLA-peptide complex containing the first or second peptide. In some embodiments, the CD8 +The TCR of the T cell binds to a class II HLA-peptide complex comprising the first or second peptide. In some embodiments, the one or more APCs comprise a first APC comprising the first peptide and a second APC comprising the second peptide. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a read-through mutation, a gene fusion mutation, and any combination thereof. In some embodiments, the first neoepitope and the second neoepitope comprise a sequence encoded by a gene in Table 1 or 2. In some embodiments, the protein is encoded by a gene in Table 1 or 2. In some embodiments, the mutation is a mutation in the second column of Table 1 or 2. In some embodiments, the protein is GATA3. In some embodiments, the first neoepitope and the second neoepitope comprise a sequence encoded by a gene in Table 34 or Table 36. In some embodiments, the protein is encoded by a gene in Table 34 or Table 36. In some embodiments, the mutation is a mutation in the second column of Table 34 or Table 36. In some embodiments, the protein is BTK. In some embodiments, the first neoepitope and the second neoepitope comprise sequences encoded by genes in Tables 40A-40D. In some embodiments, the protein is encoded by a gene in Table 3 or 35. In some embodiments, the mutation is a mutation in the second column of Table 3 or 35. In some embodiments, the protein is EGFR. In some embodiments, a single polypeptide comprises the first peptide and the second peptide, or a single polynucleotide encodes the first peptide and the second peptide. In some embodiments, the first peptide and the second peptide are encoded by sequences transcribed from the same transcription start site. In some embodiments, the first peptide is encoded by a sequence transcribed from a first transcription start site and the second peptide is encoded by a sequence transcribed from a second transcription start site.In some embodiments, a single polypeptide has a length of at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the first corresponding wild-type sequence. and a second sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a corresponding second wild-type sequence.In some embodiments, the polypeptide comprises at least 8 or 9 contiguous amino acids that have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding first wild-type sequence. The peptides comprise a first sequence and a second sequence of at least 16 or 17 consecutive amino acids that have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding second wild-type sequence. In some embodiments, the second peptide is longer than the first peptide. In some embodiments, the first peptide is longer than the second peptide. In some embodiments, the first peptide has a length of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the second peptide has a length of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids.In some embodiments, the first peptide comprises a sequence of at least 9 contiguous amino acids that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding wild-type sequence. In some embodiments, the second peptide comprises a sequence of at least 17 contiguous amino acids that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding wild-type sequence. In some embodiments, the second neoepitope is longer than the first neoepitope. In some embodiments, the first neoepitope is at least 8 amino acids in length. In some embodiments, the first neoepitope has a length of 8 to 12 amino acids. In some embodiments, the first neoepitope comprises at least 8 contiguous amino acids, wherein at least two of the 8 contiguous amino acids differ from those at the corresponding positions in the wild-type sequence. In some embodiments, the second neoepitope has a length of at least 16 amino acids. In some embodiments, the second neoepitope has a length of 16 to 25 amino acids. In some embodiments, the second neoepitope comprises at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from those at the corresponding positions in the wild-type sequence.

[0318] In some embodiments, the first peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the first neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the first neoepitope. In some embodiments, the second peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the second neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the second neoepitope. In some embodiments, the first peptide, the second peptide, or both, comprise at least one flanking sequence, wherein the at least one flanking sequence is upstream or downstream of the neoepitope. In some embodiments, at least one flanking sequence has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, at least one flanking sequence comprises a non-wild-type sequence. In some embodiments, at least one flanking sequence is an N-terminal flanking sequence. In some embodiments, at least one flanking sequence is a C-terminal flanking sequence. In some embodiments, at least one flanking sequence of a first peptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least one flanking sequence of a second peptide.In some embodiments, at least one flanking region of the first peptide differs from at least one flanking region of the second peptide. In some embodiments, at least one flanking residue comprises a mutation. In some embodiments, the first neoepitope, the second neoepitope, or both comprise at least one anchor residue. In some embodiments, at least one anchor residue of the first neoepitope is at a canonical anchor position. In some embodiments, at least one anchor residue of the first neoepitope is at a non-canonical anchor position. In some embodiments, at least one anchor residue of the second neoepitope is at a canonical anchor position. In some embodiments, at least one anchor residue of the second neoepitope is at a non-canonical anchor position. In some embodiments, at least one anchor residue of the first neoepitope differs from at least one anchor residue of the second neoepitope. In some embodiments, at least one anchor residue is a wild-type residue. In some embodiments, at least one anchor residue is a substitution. In some embodiments, the first neoepitope and / or the second neoepitope bind to an HLA protein with greater affinity than the corresponding neoepitope without the substitution. In some embodiments, the first neoepitope and / or the second neoepitope bind to an HLA protein with greater affinity than the corresponding wild-type sequence without the substitution. In some embodiments, at least one anchor residue does not comprise a mutation. In some embodiments, the first neoepitope, the second neoepitope, or both, comprise at least one anchor residue flanking region. In some embodiments, the neoepitope comprises at least one anchor residue. In some embodiments, the at least one anchor residue comprises at least two anchor residues. In some embodiments, the at least two anchor residues are separated by a separation region comprising at least one amino acid. In some embodiments, the at least one anchor residue flanking region is not within the separation region.In some embodiments, at least one anchor residue-adjacent region is both (a) and (b) downstream of a C-terminal anchor residue of the at least two anchor residues and upstream of an N-terminal anchor residue of the at least two anchor residues.

[0319] In some embodiments, the composition includes an adjuvant. In some embodiments, the composition includes one or more additional peptides, wherein the one or more additional peptides include a third neoepitope. In some embodiments, the first and / or second neoepitope binds to an HLA protein with greater affinity than the corresponding wild-type sequence. In some embodiments, the first and / or second neoepitope has a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neoepitope binds to an HLA protein with a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neoepitope binds to an HLA class I protein with a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50In some embodiments, the first and / or second neoepitope binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the mutation is not present in non-cancer cells of the subject. In some embodiments, the first and / or second neoepitope is encoded by a gene or expressed gene in cancer cells of the subject. In some embodiments, the composition comprises a first T cell comprising a first TCR. In some embodiments, the composition comprises a second T cell comprising a second TCR. In some embodiments, the first TCR comprises a non-native intracellular domain, and / or the second TCR comprises a non-native intracellular domain. In some embodiments, the first TCR is a soluble TCR, and / or the second TCR is a soluble TCR. In some embodiments, the first and / or second T cell is a cytotoxic T cell. In some embodiments, the first and / or second T cell is a gamma delta T cell. In some embodiments, the first and / or second T cells are helper T cells. In some embodiments, the first T cells are T cells stimulated, expanded, or induced with a first neoepitope, and / or the second T cells are T cells stimulated, expanded, or induced with a second neoepitope. In some embodiments, the first and / or second T cells are autologous T cells. In some embodiments, the first and / or second T cells are allogeneic T cells. In some embodiments, the first and / or second T cells are engineered T cells. In some embodiments, the first and / or second T cells are T cells of a cell line. In some embodiments, the first and / or second TCR has a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. D or IC 50The first and second peptides bind to HLA-peptide complexes at a specific site. In some aspects, provided herein is a vector comprising a polynucleotide encoding the first and second peptides described herein. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the vector comprises a self-amplifying RNA replicon, a plasmid, a phage, a transposon, a cosmid, a virus, or a virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpesvirus, a poxvirus, an alphavirus, a vaccinia virus, a hepatitis B virus, a human papillomavirus, or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, a cationic lipid, a cationic polymer, a metal nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.

[0320] In some aspects, provided herein is a pharmaceutical composition comprising a composition described herein or a vector described herein and a pharmaceutically acceptable excipient.

[0321] In some embodiments, the plurality of cells are autologous cells. In some embodiments, the plurality of APC cells are autologous cells. In some embodiments, the plurality of T cells are autologous cells. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the adjuvant is polyICLC. In some embodiments, the adjuvant is hirutonol.

[0322] In some aspects, provided herein is a method of treating cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0323] In some aspects, provided herein is a method of preventing resistance to cancer treatment, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0324] In some aspects, provided herein is a method of inducing an immune response, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0325] In some embodiments, the immune response is a humoral response. In some embodiments, the first peptide and the second peptide are administered simultaneously, separately, or sequentially. In some embodiments, the first peptide is administered sequentially after the second peptide. In some embodiments, the second peptide is administered sequentially after the first peptide. In some embodiments, the first peptide is administered sequentially after a period sufficient for the second peptide to activate T cells. In some embodiments, the second peptide is administered sequentially after a period sufficient for the first peptide to activate T cells. In some embodiments, the first peptide is administered sequentially after the second peptide to restimulate T cells. In some embodiments, the second peptide is administered sequentially after the first peptide to restimulate T cells. In some embodiments, the first peptide is administered to stimulate T cells, and the second peptide is administered after the first peptide to restimulate T cells. In some embodiments, a second peptide is administered to stimulate the T cells, and the first peptide is administered after the second peptide to restimulate the T cells.

[0326] In some embodiments, the subject has cancer, and the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is breast cancer that is resistant to anti-estrogen therapy, MSI breast cancer, metastatic breast cancer, Her2-negative breast cancer, Her2-positive breast cancer, ER-negative breast cancer, ER-positive breast cancer, PR-positive breast cancer, PR-negative breast cancer, or any combination thereof. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has breast cancer that is resistant to anti-estrogen therapy. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL that is resistant to ibrutinib therapy. In some embodiments, the CLL expresses a Bruton's tyrosine kinase with a mutation, such as a C481S mutation. In some embodiments, the subject has lung cancer that is resistant to tyrosine kinase inhibitors. In some embodiments, the lung cancer expresses an epidermal growth factor receptor (EGFR) with a mutation, such as a T790M mutation. In some embodiments, a plurality of APC cells comprising a first peptide and a plurality of APC cells comprising a second peptide are administered simultaneously, separately, or sequentially. In some embodiments, a plurality of T cells comprising a first TCR and a plurality of T cells comprising a second TCR are administered simultaneously, separately, or sequentially. In some embodiments, the method further comprises administering at least one additional therapeutic agent or therapy. In some embodiments, the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered prior to, concurrently with, or following administration of a pharmaceutical composition as described herein. peptide

[0327] In embodiments, the present disclosure provides isolated peptides comprising tumor-specific mutations from Tables 1 or 2. In embodiments, the present disclosure provides isolated peptides comprising tumor-specific mutations from Table 34. In embodiments, the present disclosure provides isolated peptides comprising tumor-specific mutations from Tables 40A-40D. These peptides and polypeptides are referred to herein as "neo-antigenic peptides" or "neo-antigenic polypeptides." As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to a polymer of amino acid residues, usually L-amino acids, connected one to the other by a peptide bond, usually between the α-amino and carboxyl groups of adjacent amino acids. Polypeptides and peptides include, but are not limited to, "mutant peptides," "neo-antigenic peptides," and "neo-antigenic peptides." Polypeptides and peptides can be of various lengths, in either their neutral (uncharged) form or in their salt form, and can be free of or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein. A peptide or polypeptide can include at least one flanking sequence. As used herein, the term "flanking sequence" refers to a fragment or region of a peptide that is not part of the epitope. Table 1 lists GATA3 neo-ORF peptides. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] Table 2 below lists exemplary selected peptides. [Table 2-1] [Table 2-2] 1 Underlined AA represents non-native AA Table 3 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] 1 Underlined AA represents non-native AA 2 Bold AA represents the native AA of the amino acid sequence encoded by the second of the two fused genes. 3 The bold and underlined AA represents a non-native AA in the amino acid sequence encoded by the second of the two fused genes due to a frameshift.

[0328] A highly common mutation in ibrutinib, a molecule that targets Bruton's tyrosine kinase (BTK) and is used to treat CLL and certain lymphomas, is a cysteine ​​to serine change at position 481 (C481S). This change results in multiple binding peptides that bind to various HLA molecules. The mutation occurs in the amino acid sequence: [ka] The mutated serine is underlined and is within the region having

[0329] Exemplary neoantigenic peptides corresponding to the C481S mutation are presented in Table 34. This table also provides a list of HLA alleles whose encoded protein products can bind to the peptide. In some embodiments, the present disclosure provides C481S neoepitopes of cancer therapeutics, such as ANGSLLNY; ANGSLLNYL; ANGSLLNYLR; EYMANGSL; EYMANGSLLN; EYMANGSLLNY; GSLLNYLR; GSLLNYLREM; ITEYMANGS; ITEYMANGSL; ITEYMANGSLL; MANGOSLNYL; MANGOSLNYLR; NGSLLNYL; NGSLLNYL; SLLNYLREMR; TEYMANGSLL; TEYMANGSLLNY; YMANGSLL; and YMANGSLLN. Tables 35 and 36 provide exemplary neoantigen candidates corresponding to other cancer-associated genetic mutations. Figure 36 provides a list of selected HLA-restricted BTK peptides for this application and the corresponding proteins encoded by HLA alleles to which the mutant BTK peptides bind or are predicted to bind. Table 37 provides a list of selected BTK peptides and the corresponding preferred proteins encoded by HLA alleles to which the peptides bind or are predicted to bind, as applicable to the context of this application. Table 34 below lists exemplary neoantigenic peptides corresponding to the C481S mutation. [Table 34] Table 35 provides exemplary neoantigen candidates corresponding to other cancer-associated genetic mutations. [Table 35-1] [Table 35-2] [Table 35-3] [Table 35-4]

Table 35-5

Table 35-6

Table 35-7

Table 35-8

Table 35-9

Table 35-10

Table 35-11

Table 35-12

Table 35-13

Table 35-14

Table 35-15

Table 35-16

Table 35-17

Table 35-18

Table 35-19

Table 35-20

Table 35-21

[0330] Exemplary mutations in the EGFR gene frequently found in various types of cancer are presented in Tables 40A-40D. The tables also provide exemplary EGFR neo-antigenic peptides. Mutations involving single amino acid substitutions frequently found in cancer are listed in Tables 40A-40C. Exemplary mutations involving deletions or deletions and insertions are presented in Table 40D. Table 40A. Exemplary EGFR point mutations and mutant peptides in cancer [Table 40-1] [Table 40-2] [Table 40-3] Table 40B. Exemplary EGFR point mutations and mutant peptides in cancer [Table 40-4] [Table 40-5] Table 40C. Exemplary EGFR point mutations and mutant peptides in cancer [Table 40-6] Table 40D. Exemplary EGFR deletion and fusion mutations in cancer [Table 40-7] In the above table, for one or more of the exemplary fusions, the sequence before the first ":" belongs to the exon sequence of the polypeptide encoded by the first gene, the sequence after the second ":" belongs to the exon sequence of the polypeptide encoded by the second gene, and the amino acids appearing between the ":" symbols are encoded by codons that are split between the exon sequence of the polypeptide encoded by the first gene and the exon sequence of the polypeptide encoded by the second gene.

[0331] However, in some embodiments, e.g., NAB:STAT6, the NAB exon is linked to the 5'UTR of STAT6, and the first amino acid that appears after the junction is the normal start codon for STAT6 (there is no frame at this site, as it is not normally translated).

[0332] AR-V7 in the table above can also be considered, in some embodiments, a splice variant of the AR gene that encodes a protein that lacks the ligand-binding domain found in the full-length AR.

[0333] In some embodiments, sequencing is used to identify tumor-specific mutations. Any suitable sequencing method, such as next-generation sequencing (NGS), can be used in accordance with the present disclosure. Third-generation sequencing methods may replace NGS in the future to accelerate the sequencing step of the method. For clarity, the term "next-generation sequencing" or "NGS" in the context of this disclosure refers to any novel high-throughput sequencing technology that randomly reads nucleic acid templates in parallel along the entire genome by breaking the genome into small pieces, as opposed to the "traditional" sequencing methodology known as Sanger chemistry. Such NGS technologies (also known as massively parallel sequencing technologies) can provide nucleic acid sequence information for the entire genome, exome, transcriptome (all transcribed sequences in the genome), or methylome (all methylated sequences in the genome) in a very short period of time, e.g., within 1-2 weeks, e.g., within 1-7 days, or even within less than 24 hours, thereby enabling single-cell sequencing approaches in principle. Several NGS platforms that are commercially available or described in the literature, such as those described in detail in WO2012 / 159643, can be used in connection with the present disclosure.

[0334] In certain embodiments, the peptides described herein may be any of a wide variety of peptides, including, but not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about The neo-antigenic peptide molecule may comprise 8, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 150, about 200, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 7,500, about 10,000 amino acids or more, and any range derivable therein. In certain embodiments, the neo-antigenic peptide molecule is equal to or less than 100 amino acids.

[0335] In some embodiments, the peptides can be from about 8 to about 50 amino acid residues in length, or from about 8 to about 30 amino acid residues in length, from about 8 to about 20 amino acid residues in length, from about 8 to about 18 amino acid residues in length, from about 8 to about 15 amino acid residues in length, or from about 8 to about 12 amino acid residues in length. In some embodiments, the peptides can be from about 8 to about 500 amino acid residues in length, or from about 8 to about 450 amino acid residues in length, from about 8 to about 400 amino acid residues in length, from about 8 to about 350 amino acid residues in length, from about 8 to about 300 amino acid residues in length, from about 8 to about 250 amino acid residues in length, from about 8 to about 200 amino acid residues in length, from about 8 to about 150 amino acid residues in length, from about 8 to about 100 amino acid residues in length, from about 8 to about 50 amino acid residues in length, or from about 8 to about 30 amino acid residues in length.

[0336] In some embodiments, peptides may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues in length. In some embodiments, peptides may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues in length. In some embodiments, the peptides may be at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues in length. In some embodiments, the peptides may be at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues in length.

[0337] In some embodiments, the peptides have an overall length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0338] In some embodiments, the peptide has an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

[0339] Longer peptides can be designed in several ways. In some embodiments, when HLA-binding peptides are predicted or known, the longer peptides include (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product, or (2) a concatenation of some or all of the binding peptides, each with an extension sequence. In other embodiments, when sequencing reveals a long (>10 residue) neoepitope sequence present in the tumor (e.g., due to frameshift, readthrough, or intron inclusion resulting in a novel peptide sequence), the longer peptide can consist of the entire stretch of novel tumor-specific amino acids, either a single longer peptide or several overlapping longer peptides. In some embodiments, the use of longer peptides is presumed to allow endogenous processing by patient cells, which can result in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, with the peptides overlapping and tiling across the long neoantigenic peptide.

[0340] In some embodiments, the peptide may have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the peptide may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or greater. In some embodiments, the peptide may have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or less.

[0341] In some embodiments, the peptides described herein may be dissolved, lyophilized, or in crystalline form. In some embodiments, the peptides described herein may be synthetically prepared by recombinant DNA technology or chemical synthesis, or isolated from natural sources such as natural tumors or pathogenic organisms. Neoepitopes may be synthesized individually or may be conjugated directly or indirectly within the peptide. While the peptides described herein may be substantially free of other naturally occurring host cell proteins and their fragments, in some embodiments, the peptides may be synthetically conjugated to native fragments or particles.

[0342] In some embodiments, the peptides described herein can be prepared in a variety of ways. In some embodiments, peptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart & Young, Solid Phase Peptide Synthesis, 2d.Ed., Pierce Chemical Co., 1984. Furthermore, chemical ligation can be used to join individual peptides to generate longer peptides, which are also within the scope of the present disclosure.

[0343] Alternatively, recombinant DNA technology can be utilized in which a nucleotide sequence encoding the peptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression. These procedures are generally known in the art, as generally described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Thus, recombinant peptides comprising one or more neo-antigenic peptides described herein can be used to present appropriate T cell epitopes.

[0344] In some embodiments, the peptide is encoded by a gene with a point mutation that results in an amino acid substitution of the native peptide. In some embodiments, the peptide is encoded by a gene with a point mutation that results in a frameshift mutation. A frameshift occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in translation of a non-native protein sequence. Different mutations in a gene can acquire the same altered reading frame. In some embodiments, the peptide is encoded by a gene with a mutation that results in a fusion polypeptide, an in-frame deletion, an insertion, expression of an endogenous retroviral polypeptide, and tumor-specific overexpression of the polypeptide. In some embodiments, the peptide is encoded by a fusion of a first gene with a second gene. In some embodiments, the peptide is encoded by an in-frame fusion of a first gene with a second gene. In some embodiments, the peptide is encoded by a fusion of a first gene with an exon of a splice variant of the first gene. In some embodiments, the peptide is encoded by a fusion of a first gene and a cryptic exon of the first gene. In some embodiments, the peptide is encoded by a fusion of a first gene and a second gene, wherein the peptide comprises an amino acid sequence encoded by an out-of-frame sequence resulting from the fusion.

[0345] In some aspects, the present disclosure provides compositions comprising at least two or more peptides. In some embodiments, the compositions described herein contain at least two distinct peptides. In some embodiments, the compositions described herein contain a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope. In some embodiments, the first and second peptides are derived from the same protein. The at least two distinct peptides may differ in length, amino acid sequence, or both. The peptides may be derived from any protein known or found to contain a tumor-specific mutation. In some embodiments, the compositions described herein include a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, wherein the first peptide is different from the second peptide, the first neoepitope contains a mutation, and the second neoepitope contains the same mutation. In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a first region of a protein and a second peptide comprising a second neoepitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide differs from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, a gene fusion mutation, and any combination thereof.

[0346] In some embodiments, peptides can be derived from proteins with substitution mutations, such as KRAS G12C, G12D, G12V, Q61H, or Q61L mutations, NRAS Q61K or Q61R mutations, BTK C481S mutations, or EGFR S492R or EGFR T490M mutations. The substitutions can be located anywhere along the length of the peptide. For example, the substitutions can be located in the N-terminal third of the peptide, the central third of the peptide, or the C-terminal third of the peptide. In other embodiments, the substituted residues can be located 2-5 residues away from the N-terminus or 2-5 residues away from the C-terminus. Peptides can also be derived from tumor-specific insertion mutations, in which case the peptide includes one or more or all of the inserted residues.

[0347] In some embodiments, the first peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the first neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the first neoepitope. In some embodiments, the second peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the second neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the second neoepitope.

[0348] In some aspects, the present disclosure provides compositions comprising a single polypeptide comprising a first peptide and a second peptide, or a single polynucleotide encoding the first peptide and the second peptide. In some embodiments, the compositions provided herein comprise one or more additional peptides, wherein the one or more additional peptides comprise a third neoepitope. In some embodiments, the first peptide and the second peptide are encoded by sequences transcribed from the same transcription start site. In some embodiments, the first peptide is encoded by a sequence transcribed from a first transcription start site, and the second peptide is encoded by a sequence transcribed from a second transcription start site. In some embodiments, the polypeptide has a length of at least 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding wild-type sequence. and a second sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding wild-type sequence.In some embodiments, the polypeptide comprises at least 8 or 9 contiguous amino acids that have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence. a first sequence and a second sequence of at least 16 or 17 contiguous amino acids that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding wild-type sequence.

[0349] In some embodiments, the second peptide is longer than the first peptide. In some embodiments, the first peptide is longer than the second peptide. In some embodiments, the first peptide is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids in length. In some embodiments, the second peptide has a length of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the first peptide comprises a sequence of at least 9 contiguous amino acids that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding wild-type sequence. In some embodiments, the second peptide comprises a sequence of at least 17 contiguous amino acids having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence.

[0350] In some embodiments, the first peptide, the second peptide, or both, comprise at least one flanking sequence, and the at least one flanking sequence is upstream or downstream of the neoepitope. In some embodiments, the at least one flanking sequence has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the at least one flanking sequence comprises a non-wild-type sequence. In some embodiments, at least one flanking sequence is an N-terminal flanking sequence. In some embodiments, at least one flanking sequence is a C-terminal flanking sequence. In some embodiments, at least one flanking sequence of a first peptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one flanking sequence of a second peptide. In some embodiments, at least one flanking region of the first peptide differs from at least one flanking region of the second peptide, hi some embodiments, at least one flanking residue comprises a mutation.

[0351] In some embodiments, the neo-antigenic peptide with flanking sequences has the formula (N-terminal Xaa): N -(Xaa BTK ) P -(Xaa-C terminus) C (In the formula, (Xaa BTK ) Pis a mutant BTK peptide sequence comprising at least 8 consecutive amino acids of a mutant BTK protein; P is an integer greater than 7; N is (i) 0 or (ii) an integer greater than 2; (N-terminal Xaa) N is any amino acid sequence heterologous to the mutant protein; C is (i) 0 or (ii) an integer greater than 2; (Xaa-C terminus) C is any amino acid sequence heterologous to the mutant BTK protein; and both N and C are not 0).

[0352] In some embodiments, the neo-antigenic peptide with flanking sequences has the formula (N-terminal Xaa): N -(Xaa EGFR ) P -(Xaa-C terminus) C (In the formula, (Xaa EGFR ) P is a mutant EGFR peptide sequence comprising at least 8 consecutive amino acids of a mutant EGFR protein; P is an integer greater than 7; N is (i) 0 or (ii) an integer greater than 2; (N-terminal Xaa) N is any amino acid sequence heterologous to the mutant EGFR protein; C is (i) 0 or (ii) an integer greater than 2; (Xaa-C terminus) C is any amino acid sequence heterologous to the mutant EGFR protein; and both N and C are not 0).

[0353] In some embodiments, the peptide comprises a neoepitope sequence comprising at least one mutant amino acid. In some embodiments, the peptide comprises a neoepitope sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutant amino acids. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid.In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid.

[0354] In some embodiments, the peptide comprises a neo-antigenic peptide sequence shown in Table 1 or 2. In some embodiments, the peptide comprises a neo-epitope sequence shown in Table 1 or 2. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutant amino acids (underlined amino acids) as shown in Table 1 or 2. In some embodiments, the peptide comprises a neo-antigenic peptide sequence shown in Table 34 or 36. In some embodiments, the peptide comprises a neo-epitope BTK sequence shown in Table 34 or 36. In some embodiments, the peptide comprises a neoepitope sequence comprising at least one mutant amino acid as shown in Table 34 or 36. In some embodiments, the peptide comprises a neoepitope sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutant amino acids. In some embodiments, the peptide comprises a neoepitope sequence comprising at least one mutant amino acid (underlined amino acid) and at least one bolded amino acid as shown in Table 1 or 2. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids, as shown in Table 1 or 2.In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid.

[0355] In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid, as shown in Table 34 or 36. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid, as shown in Table 34 or 36.

[0356] In some embodiments, the peptide comprises a neo-antigenic peptide sequence shown in Tables 40A-40D, 32, or 3A-3D. In some embodiments, the peptide comprises a neo-epitope EGFR sequence shown in Tables 40A-40D. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least one mutant amino acid as shown in Tables 40A-40D. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more mutant amino acids (e.g., the underlined amino acids in any one of Tables 40A-40D). In some embodiments, the EGFR peptide comprises a neoepitope sequence that includes at least one mutant amino acid shown in bold as shown in Table 40D. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids as shown in Tables 40A-40D. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (e.g., an underlined amino acid in Table 40C), as shown in Tables 40A-40D, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid.In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid, as shown in Tables 40A-40D. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids upstream of the at least one mutant amino acid and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-mutated amino acids downstream of the at least one mutant amino acid, as shown in Tables 40A-40D.

[0357] In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid and a sequence upstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the peptide has at least one mutant amino acid and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence. and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.

[0358] In some embodiments, the peptide comprises at least one mutant amino acid and, upstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 16 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the protein. In some embodiments, the peptide comprises at least one mutant amino acid and, downstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 16 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the protein.In some embodiments, the peptide comprises at least one mutant amino acid and, upstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155% or more of a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive amino acids and a sequence downstream of the at least one mutant amino acid that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 16 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids with 3%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0359] In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and a sequence upstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a sequence similar to the corresponding wild-type sequence. and a neoepitope sequence derived from a protein comprising a sequence having 100% sequence identity and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.

[0360] In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and, upstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% or more of a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and a sequence downstream of the at least one mutant amino acid that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% or more of a sequence similar to the corresponding wild-type sequence. and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 1 or 2 and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151% nucleotides, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 1 , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more a sequence containing many consecutive amino acids and downstream of this at least one mutant amino acid, which is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% of the corresponding wild-type sequence; and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the protein.

[0361] In some embodiments, the BTK peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (underlined amino acid) as set forth in Table 34 or 36 and a sequence upstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid as set forth in Table 34 or 36 and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the peptide comprises at least one mutant amino acid as set forth in Table 34 or 36 and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding wild-type sequence. and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.

[0362] In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 34 or 36 and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the peptide comprises at least one mutant amino acid as set forth in Table 34 or 36 and, downstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Table 34 or 36 and, upstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more sequences that share 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and a sequence containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510%, 1520%, 1530%, 1540%, 1550%, 1560%, 1570%, 1580%, 1590%, 1610%, 1620%, 1630%, 1640%, 1650%, 166 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the protein.

[0363] Exemplary neoantigenic peptides corresponding to the C481S mutation are presented in Table 34. This table also provides a list of HLA alleles whose encoded protein products can bind to the peptide. In some embodiments, the peptide comprising the C481S mutation: [ka] In some embodiments, a peptide comprising a BTK mutation comprises the neoepitope sequence of ANGSLLNY. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of ANGSLLNYL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of ANGSLLNYLR. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of EYMANGSL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of EYMANGSLLN. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of EYMANGSLLNY. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of GSLLNYLR. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of GSLLNYLREM. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of ITEYMANGS. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of ITEYMANGSL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of ITEYMANGSLL. MANGSLLNYL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of MANGSLLNYLR. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of NGSLLNYL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of NGSLLNYL. In some embodiments, a peptide comprising a C481S BTK mutation comprises the neoepitope sequence of SLLNYLREMR. In some embodiments, a peptide comprising the C481S BTK mutation comprises a neoepitope sequence of TEYMANGSLL;TEYMANGSLLNY. In some embodiments, a peptide comprising the C481S BTK mutation comprises a neoepitope sequence of YMANGSLL.

[0364] In some embodiments, the EGFR peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (underlined amino acid) as shown in Tables 40A-40D and a sequence upstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (underlined amino acid) as shown in Tables 40A-40D and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the peptide comprises at least one mutant amino acid as set forth in Tables 40A-40D and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding wild-type sequence. and a sequence downstream of the at least one mutant amino acid having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the corresponding wild-type sequence.

[0365] In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Tables 40A-40D and, upstream of the at least one mutant amino acid, at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 1 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Tables 40A-40D and, downstream of the at least one mutant amino acid, a sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 15 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) as shown in Tables 40A-40D and a sequence upstream of the at least one mutant amino acid that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more sequences that share 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and a sequence containing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 1510%, 1520%, 1530%, 1540%, 1550%, 1560%, 1570%, 1580%, 1590%, 1610%, 1620%, 1630%, 1640%, 1650%, 166 and a sequence comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the protein.

[0366] In some embodiments, the peptide comprising the EGFR T790M mutation is [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the mutant EGFR peptide comprising the EGFR T790M mutation comprises the neoepitope sequence: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] In some embodiments, the peptide comprising the EGFR T790M mutation comprises a neoepitope sequence of: [ka] It contains the neoepitope sequence of

[0367] In some embodiments, the peptide comprising the EGFR S492R mutation is [ka] In some embodiments, the peptide comprising the EGFR S492R mutation comprises the sequence: [ka] It contains the neoepitope sequence of

[0368] In some embodiments, the EGFR neopeptide is selected from Tables 40A-40D.

[0369] In some embodiments, peptides comprising a deletion mutation of EGFR, such as a deletion of G in EGFRvIII (an internal deletion); [ka] teeth, [ka] It contains the neoepitope sequence of

[0370] In some embodiments, the sequence: [ka] The peptide containing the mutation shown in [ka] In some embodiments, the neoepitope sequence comprises the sequence: [ka] The peptide containing the mutation shown in [ka] In some embodiments, the neoepitope sequence comprises the sequence: [ka] The peptide containing the mutation shown in [ka] In some embodiments, the neoepitope sequence comprises the sequence: [ka] The peptide containing the mutation shown contains the neoepitope sequence of . Peptide Modification

[0371] In some embodiments, the present disclosure includes modified peptides. Modifications can include covalent chemical modifications that do not alter the primary amino acid sequence of the antigenic peptide itself. Modifications can result in peptides with desired properties, such as increased in vivo half-life, increased stability, reduced clearance, altered immunogenicity or allergenicity, enabling the production of specific antibodies, cellular targeting, antigen uptake, antigen processing, HLA affinity, HLA stability, or antigen presentation. In some embodiments, the peptides can include one or more sequences that enhance epitope processing and presentation by APCs, for example, to generate an immune response.

[0372] In some embodiments, peptides can be modified to achieve desired properties. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a helper T cell response. In some embodiments, the immunogenic peptide / helper T conjugate can be linked by a spacer molecule. In some embodiments, the spacer comprises a relatively small, neutral molecule that is substantially uncharged under physiological conditions, such as an amino acid or amino acid mimetic. The spacer can be selected from, for example, an Ala spacer, a Gly spacer, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that the optional spacer need not be composed of the same residues and can therefore be a hetero- or homo-oligomer. The neo-antigenic peptide can be linked to the helper T peptide at either the amino or carboxy terminus of the peptide, either directly or via a spacer. The amino terminus of either the neo-antigenic peptide or the helper T peptide can be acylated. Examples of helper T peptides include tetanus toxoid residues 830-843, influenza residues 307-319, and malaria circumsporozoite residues 382-398 and residues 378-389.

[0373] The peptide sequences of the present disclosure can be modified as desired by changes at the DNA level, specifically by mutating preselected bases in the DNA encoding the peptide to generate codons that will be translated into the desired amino acids.

[0374] In some embodiments, the peptides described herein can contain substitutions that modify the physical properties (e.g., stability or solubility) of the resulting peptide. For example, peptides can be modified by substitution of cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​has a tendency to form disulfide bonds, structurally altering the peptide sufficiently to reduce its binding capacity. Substitution of C with α-aminobutyric acid not only alleviates this problem, but in certain cases may actually improve binding and cross-linking capacity. Substitution of cysteine ​​with α-aminobutyric acid can occur at any residue in the neoantigenic peptide, for example, at either anchor or non-anchor positions of an epitope or analog within the peptide, or at other positions in the peptide.

[0375] Peptides can also be modified by lengthening or shortening the amino acid sequence of the compound, for example, by adding or deleting amino acids. Peptides or analogs can also be modified by changing the order or composition of certain residues. Those skilled in the art will understand that certain amino acid residues essential for biological activity, such as those in essential contact sites or conserved residues, may generally be altered with deleterious effects on biological activity. Non-essential amino acid chains are not necessarily limited to those naturally occurring in proteins, such as L-α-amino acids, but can also include D-isomers, unnatural amino acids such as β-γ-δ-amino acids, and many derivatives of L-α-amino acids.

[0376] In some embodiments, peptides can be modified using a series of peptides with single amino acid substitutions to determine the effects of charge, hydrophobicity, and the like on HLA binding. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions can be made along the length of the peptide to display different sensitivity patterns for various HLA molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral molecules such as Ala, Gly, Pro, or similar residues can be utilized. Substitutions can be homo- or hetero-oligomers. The number and type of residues substituted or added depend on the required spacing between essential contact points and the specific functional characteristics desired (e.g., hydrophobicity vs. hydrophilicity). Such substitutions can also achieve increased binding affinity for HLA molecules or T cell receptors compared to the affinity of the parent peptide. In any case, such substitutions should utilize amino acid residues or other molecular fragments selected to avoid, for example, steric and charge hindrances that might interfere with binding. Amino acid substitutions are typically single residue substitutions. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final peptide.

[0377] In some embodiments, the peptides described herein contain amino acid mimetics or unnatural amino acid residues, such as D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, -2, 3-, or 4-pyrenylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-alanine; The unnatural amino acid residues may include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides with various amino acid mimetics or unnatural amino acid residues may have increased in vivo stability. Such peptides may also have improved shelf life or manufacturing characteristics.

[0378] In some embodiments, the peptides described herein can be prepared by terminal NH2 acylation, e.g., alkanoyl (C1-C 20) or thioglycolyl acetylation, terminal carboxylamidation, e.g., ammonia, methylalanine, etc. In some embodiments, these modifications can provide sites for linkage to a support or other molecule. In some embodiments, the peptides described herein can include modifications, such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactants, e.g., lipids, or the peptides described herein can be chemically modified, e.g., acetylation, etc. Additionally, bonds in the peptides can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0379] In some embodiments, the peptides described herein can include a carrier such as those known in the art, e.g., thyroglobulin, albumin, e.g., human serum albumin, tetanus toxoid, polyamino acid residues, e.g., poly-L-lysine and poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, etc.

[0380] Peptides can be further modified to contain additional chemical moieties not normally part of proteins. These derivatized moieties can improve solubility, biological half-life, protein absorption, or binding affinity. These moieties can also reduce or eliminate any undesirable side effects of the peptide. A summary of these moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, neoantigenic peptides with desired activity can be modified as needed to enhance or at least retain substantially all of the biological activity of the unmodified peptide, which binds to the desired HLA molecule and activates appropriate T cells, while also providing certain desired attributes, such as improved pharmacological properties. For example, peptides can be subject to various changes, such as either conservative or non-conservative substitutions, which may provide certain advantages in their use, such as improved HLA binding. Such conservative substitutions can involve replacing an amino acid residue with another amino acid residue that is biologically and / or chemically similar, for example, replacing one hydrophobic residue with another hydrophobic residue, or one polar residue with another polar residue. The effect of single amino acid substitutions can also be explored using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, for example, as described in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0381] In some embodiments, the peptides described herein can be conjugated to large, slowly metabolized macromolecules, such as proteins; polysaccharides, e.g., sepharose, agarose, cellulose, cellulose beads; polymeric amino acids, e.g., polyglutamic acid, polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins, e.g., toxins from diphtheria, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells.

[0382] Modifications to the peptide may include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation, HESylation, recombinant PEG mimics, Fc fusion, albumin fusion, nanoparticle conjugation, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimetics, or addition of unnatural amino acids.

[0383] Glycosylation can affect the physical properties of proteins and may also be important in terms of protein stability, secretion, and intracellular localization. Proper glycosylation can be important for biological activity. Indeed, some genes from eukaryotes, when expressed in bacteria (e.g., E. coli) that lack the intracellular processes for glycosylation of proteins, produce proteins with little or no activity due to their lack of glycosylation, and these proteins are recovered. Addition of glycosylation sites can be achieved by altering the amino acid sequence. Modifications to peptides or proteins can be made, for example, by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type can differ. One type of sugar that is often found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is typically the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, can confer acidic properties to glycoproteins. Embodiments of the present disclosure include the production and use of N-glycosylation variants. Carbohydrate removal can be accomplished chemically or enzymatically, or by substituting the codon encoding the glycosylated amino acid residue. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exoglycosidases.

[0384] Additional suitable components and molecules for conjugation include, for example, molecules for targeting to the lymphatic system, thyroglobulin; albumins, such as human serum albumin (HAS); tetanus toxoid; diphtheria toxoid; polyamino acids, such as poly(D-lysine:D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.

[0385] Another type of modification is the conjugation (e.g., linkage) of one or more additional components or molecules, such as another protein (e.g., a protein having an amino acid sequence heterologous to that of the protein of interest) or a carrier molecule, at the N-terminus and / or C-terminus of the polypeptide sequence. Thus, exemplary polypeptide sequences can be provided as conjugates with another component or molecule. In some embodiments, fusion of albumin to a peptide or protein of the present disclosure can be achieved, for example, by genetic engineering such that DNA encoding HSA, or a fragment thereof, is joined to DNA encoding one or more polypeptide sequences. A suitable host can then be transformed with the fusion nucleotide sequence, e.g., in the form of a suitable plasmid, or such a fusion nucleotide sequence can be transfected into the host to express the fusion polypeptide. Expression can be achieved in vitro, e.g., from prokaryotic or eukaryotic cells, or in vivo, e.g., from transgenic organisms. In some embodiments of the present disclosure, the fusion protein is expressed in a mammalian cell system, e.g., a CHO cell system. Furthermore, albumin itself can be modified to extend its circulating half-life. The fusion of modified albumin to one or more polypeptides can be achieved by the above-mentioned genetic engineering techniques or by chemical conjugation, and the resulting fusion molecule has a half-life that exceeds that of fusion with unmodified albumin (see, for example, WO2011 / 051489).Some albumin binding strategies have been developed as an alternative to direct fusion, including albumin binding via conjugated fatty acid chains (acylation).Because serum albumin is a transport protein for fatty acids, these natural ligands with albumin binding activity have been used to extend the half-life of small molecule protein therapeutics.

[0386] Additional candidate components and molecules for conjugation include those suitable for isolation or purification.Non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules that constitute solid supports, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.Conjugates can be separated by charge differences using purification methods such as cation exchange chromatography, which effectively separates conjugates into their various molecular weights.The contents of the fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectrometry, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.

[0387] In some embodiments, the amino or carboxyl terminus of a peptide or protein sequence of the present disclosure can be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, thus potentially requiring less frequent administration of biopharmaceutical products. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and released back into the circulation, resulting in longer circulation. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. More recent Fc fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc fusion conjugates.

[0388] The present disclosure contemplates the use of other modifications of peptides, currently known or developed in the future, to improve one or more properties.One such method for extending the circulating half-life of the peptides of the present disclosure, increasing stability, reducing clearance, or changing immunogenicity or allergenicity comprises modifying the peptide sequence by hydroxyethyl starch modification, which utilizes hydroxyethyl starch derivatives linked to other molecules to modify the properties of the molecule.Various aspects of hydroxyethyl starch modification are described, for example, in US Patent Application Publication No. 2007 / 0134197 and US Patent Application Publication No. 2006 / 0258607.

[0389] Peptide stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, for example, Verhoef, et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). The half-life of the peptides described herein is conveniently determined using a 25% human serum (v / v) assay. The protocol is as follows: pooled human serum (type AB, non-heat-inactivated) is disrupted by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol. The cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatographic conditions.

[0390] Problems associated with short plasma half-lives or vulnerability to protease degradation can be overcome by various modifications, including conjugating or linking the peptide or protein sequence to any of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene (e.g., usually via a linking moiety that is covalently attached to both the protein and the nonproteinaceous polymer, e.g., PEG). Such PEG-conjugated biomolecules have the potential to provide better physical and thermal stability, protection from vulnerability to enzymatic degradation, improved solubility, and longer shelf life. They have been shown to have clinically useful properties, including reduced circulating half-life and clearance in vivo, reduced immunogenicity and antigenicity, and reduced toxicity.

[0391] PEG suitable for conjugation to polypeptide or protein sequences is generally water soluble at room temperature and has the general formula R—(O—CH—CH) n-OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has 1 to 8 carbons. PEG conjugated to a polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure. The present disclosure also contemplates compositions of conjugates in which the PEGs have different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates in which n=1, 2, 3, and 4. In some compositions, the percentage of conjugates in which n=1 is 18-25%, the percentage of conjugates in which n=2 is 50-66%, the percentage of conjugates in which n=3 is 12-16%, and the percentage of conjugates in which n=4 is 5% or less. Such compositions can be produced by reaction conditions and purification methods known in the art, for example, cation exchange chromatography can be used to separate the conjugates, and then fractions containing, for example, conjugates with a desired number of PEGs attached are identified, which fractions are purified, free of unmodified protein sequences, and free of conjugates with other numbers of PEGs attached.

[0392] PEG can be attached to the peptides or proteins of the present disclosure via a terminal reactive group ("spacer"). The spacer is, for example, a terminal reactive group that mediates the attachment of PEG to one or more free amino or carboxyl groups of the polypeptide sequence. PEGs having a spacer that can be attached to free amino groups include N-hydroxysuccinimide PEG, which can be prepared by activating the succinic acid ester of PEG with N-hydroxysuccinimide. Another activated PEG that can be attached to free amino groups is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting PEG monomethyl ether with cyanuric chloride. Activated PEGs that can be attached to free carboxyl groups include polyoxyethylenediamine.

[0393] Conjugation of one or more of the peptide or protein sequences of the present disclosure to PEG having a spacer can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out in solution using a molar ratio of reagents to peptide / protein of 4:1 to 30:1, at a pH of 5 to 10, at a temperature of 4°C to room temperature, for 30 minutes to 20 hours. Reaction conditions can be selected to direct the reaction primarily to produce the desired degree of substitution. Generally, low temperatures, low pH (e.g., pH = 5), and short reaction times tend to decrease the number of PEGs attached, whereas high temperatures, medium to high pH (e.g., pH > 7), and longer reaction times tend to increase the number of PEGs attached. The reaction can be terminated using a variety of means known in the art. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing, e.g., at -20°C.

[0394] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the properties of PEG (e.g., improved serum half-life) while also conferring several additional advantageous properties. For example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser, and Thr) that can form extended conformations similar to PEG can be produced that are already recombinantly fused to the peptide or protein drug of interest (e.g., Amunix's XTEN technology; Mountain View, CA). This eliminates the need for an additional conjugation step during the manufacturing process. Furthermore, the side chain composition of the polypeptide chain can be controlled using established molecular biology techniques, thereby enabling optimization of immunogenicity and manufacturing properties. Neoepitope

[0395] Neoepitopes include neoantigenic determinants of neoantigenic peptides or polypeptides that are recognized by the immune system. Neoepitopes refer to epitopes that are absent from reference non-diseased cells, e.g., non-cancerous cells or germline cells, but are found in diseased cells, e.g., cancer cells. This includes situations where the corresponding epitope is found in normal non-diseased cells or germline cells, but one or more mutations in diseased cells, e.g., cancer cells, result in an altered sequence of the epitope, creating a neoepitope. The term "neoepitope" is used interchangeably herein with "tumor-specific neoepitope" to refer to a series of residues, usually L-amino acids, connected to each other by peptide bonds, usually between the alpha-amino and carboxyl groups of adjacent amino acids. Neoepitopes can be of various lengths, either in their neutral (uncharged) form or in salt form, and can be free of or contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein. The present disclosure provides isolated neoepitopes comprising tumor-specific mutations from Tables 1 or 2. The present disclosure also provided exemplary isolated neoepitopes comprising tumor-specific mutations from Table 34. The present disclosure also provides exemplary isolated neoepitopes comprising tumor-specific mutations from Tables 40A-40D and Tables 3A-3D.

[0396] In some embodiments, the neoepitopes described herein for HLA class I are 13 residues or less in length, typically consisting of between about 8 and about 12 residues, particularly 9 or 10 residues. In some embodiments, the neoepitopes described herein for HLA class II are 25 residues or less in length, typically consisting of between about 16 and about 25 residues.

[0397] In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, wherein the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation. In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a first region of a protein and a second peptide comprising a second neoepitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide is different from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readout mutation, a gene fusion mutation, and any combination thereof.

[0398] In some embodiments, the first neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the first neoepitope binds to a CD8 + In some embodiments, the first neoepitope activates CD4 T cells. + In some embodiments, the second neoepitope activates CD4 T cells. + In some embodiments, the second neoepitope activates CD8 T cells. + In some embodiments, CD4 +The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + T cell TCR is class I In some embodiments, the peptide binds to the HLA-peptide complex. + The TCR of the T cell binds to a class I HLA-peptide complex. In some embodiments, a composition comprising the neoantigenic C481S BTK peptide comprises a first BTK neoepitope and a second BTK neoepitope. In some embodiments, the first BTK neoepitope comprises a neoepitope selected from Table 34. In some embodiments, the second BTK neoepitope comprises a neoepitope selected from Table 34.

[0399] In some embodiments, a first mutant BTK peptide sequence selected from Table 34 binds, or is predicted to bind, to a protein encoded by an HLA allele listed in Table 34 corresponding to the respective peptide (left column to right column).

[0400] In some embodiments, the composition comprising a neoantigenic EGFR peptide comprises a first EGFR neoepitope and a second EGFR neoepitope. In some embodiments, the first EGFR neoepitope comprises a neoepitope selected from Tables 40A-40D. In some embodiments, the second EGFR neoepitope comprises a neoepitope selected from Tables 40A-40D.

[0401] In some embodiments, the first mutant EGFR neoepitope is selected from the group consisting of STVQLIMQL, LIMQLMPF, LTSTVQLIM, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM and VQLIMQLM.

[0402] In some embodiments, the first mutant EGFR peptide sequence selected from the group consisting of STVQLIMQL, LIMQLMPF, LTSTVQLIM, TVQLIMQL, TSTVQLIMQL, TVQLIMQLM, and VQLIMQLM is an HLA-A68:01 allele, an HLA-B15:02 allele, an HLA-A25:01 allele, an HLA-B57:03 allele, an HLA-B57:04 allele, an HLA-B57:05 allele, an HLA-B57:06 allele, an HLA-B57:07 allele, an HLA-B57:08 allele, an HLA-B57:09 allele, an HLA-B57:01 allele, an HLA-B57:02 allele, an HLA-B57:03 allele, an HLA-B57:04 allele, an HLA-B57:05 allele, an HLA-B57:06 allele, an HLA-B57:07 allele, an HLA-B57:08 allele, an HLA-B57:09 allele, an HLA-B57:01 allele, an HLA-B57:01 allele, an HLA-B57:02 allele, an HLA-B57:03 ... HLA-C12:02 allele, HLA-C03:02 allele, and HLA-A26:01 allele, HLA-C12:03 allele, HLA-C06:02 allele, HLA-C03:03, HLA-B52:01 allele, HLA-A30:01 allele, HLA-C02:02 allele, HLA-C12:03 allele, HLA-A11:01 allele, HLA-A32:01 allele , HLA-A02:04 allele, HLA-B15:09 allele, HLA-C17:01 allele, HLA-C03:04 allele, HLA-B08:01 allele, HLA-A01:01 allele, HLA-B42:01 allele, HLA-B57:01 allele, HLA-B14:02 allele, HLA-B37:01 allele, HLA-B36:01 allele, HLA-B38:01 allele and / or HLA-C03:03 allele, HLA-B14:02 allele, HLA-B37:01 allele, HLA-A02:03 allele, HLA-B58:02 allele, HLA-C08:01 allele, HLA-B35:01 allele, HLA-B40:01 allele, and / or HLA-B35:03 allele. Table 41 provides a list of exemplary HLA alleles that encode HLA proteins that can or are predicted to bind EGFR neo-antigenic peptides. [Table 41-1] [Table 41-2] Tables 42Ai, 42Aii and 42B show EGFR neoepitopes with predicted HLA subtype specificity. Tables 5Ai, 5Aii and 5B show EGFR neoepitopes with predicted HLA subtype specificity. Table 42Ai [Table 42-1] Table 42Aii [Table 42-2] [Table 42-3] Table 42B [Table 42-4]

[0403] In some embodiments, the first neoepitope and the second neoepitope are different epitopes. In some embodiments, the second neoepitope is longer than the first neoepitope. In some embodiments, the first neoepitope is at least 8 amino acids in length. In some embodiments, the first neoepitope is 8 to 12 amino acids in length. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, wherein at least one of the 8 contiguous amino acids differs from the corresponding position in the wild-type sequence. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, wherein at least two of the 8 contiguous amino acids differ from the corresponding position in the wild-type sequence. In some embodiments, the second neoepitope is at least 16 amino acids in length. In some embodiments, the second neoepitope is 16 to 25 amino acids in length. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, wherein at least one of the 16 contiguous amino acids differs from that at the corresponding position in the wild-type sequence. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from that at the corresponding position in the wild-type sequence.

[0404] In some embodiments, the neoepitope comprises at least one anchor residue. In some embodiments, the first neoepitope, the second neoepitope, or both comprise at least one anchor residue. In one embodiment, at least one anchor residue of the first neoepitope is at a canonical anchor position or a non-canonical anchor position. In another embodiment, at least one anchor residue of the second neoepitope is at a canonical anchor position or a non-canonical anchor position. In yet another embodiment, at least one anchor residue of the first neoepitope is different from at least one anchor residue of the second neoepitope.

[0405] In some embodiments, at least one anchor residue is a wild-type residue. In some embodiments, at least one anchor residue is a substitution. In some embodiments, at least one anchor residue does not include a mutation.

[0406] In some embodiments, the first or second neoepitope, or both, comprises at least one anchor residue-adjacent region. In some embodiments, the neoepitope comprises at least one anchor residue. In some embodiments, the at least one anchor residue comprises at least two anchor residues. In some embodiments, the at least two anchor residues are separated by a separation region comprising at least one amino acid. In some embodiments, the at least one anchor residue-adjacent region is not within the separation region. In some embodiments, the at least one anchor residue-adjacent region is (a) upstream of the N-terminal anchor residue of the at least two anchor residues, (b) downstream of the C-terminal anchor residue of the at least two anchor residues, or both (a) and (b). In some embodiments, the second neopeptide is selected from Table 34.

[0407] In some embodiments, the second neoepitope comprises a mutation T790M. In some embodiments, the second neoepitope comprises an EGFR T790M mutation: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neopeptide comprising the EGFR T790M mutation comprises the neoepitope sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the neoepitope sequence: [ka] In some embodiments, the second neoepitope comprising the EGFR T790M mutation comprises the sequence: [ka] Contains an array of

[0408] In some embodiments, the second neoepitope comprises an EGFR S492R mutation. In some embodiments, the peptide comprising the EGFR S492R mutation comprises: [ka] It contains the neoepitope sequence of

[0409] In some embodiments, the neoepitope sequence is [ka] A second EGFR neoepitope comprising a deletion mutation of EGFR, such as the deletion of G in EGFRvIII (an internal deletion).

[0410] In some embodiments, the neoepitope sequence is [ka] is an array: [ka] In some embodiments, the second neoepitope sequence comprises a mutation as set forth in [ka] In some embodiments, the second neoepitope sequence is: [ka] In some embodiments, the second neoepitope sequence is: [ka] is.

[0411] In some embodiments, the second neopeptide is selected from Table 35 or Tables 3A-3D.

[0412] In some embodiments, the neoepitope binds to an HLA protein (e.g., HLA class I or HLA class II). In some embodiments, the neoepitope binds to an HLA protein wi...

Claims

1. 1. A method for preparing a population of T cells comprising a T cell receptor (TCR) specific for a peptide:MHC complex, comprising: the peptide of the peptide:MHC complex is a variant GATA3 peptide sequence comprising at least 8 consecutive amino acids of PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); the method comprising contacting a population of immune cells with a polypeptide comprising the mutant GATA3 peptide sequence or expressing the polypeptide comprising the mutant GATA3 peptide sequence in the population of immune cells; The method, wherein the population of immune cells is a population of immune cells depleted of cells expressing CD25 and CD14 and a population of T cells.

2. The method described in claim 1, wherein expressing the polypeptide comprises contacting the population of immune cells with a polynucleotide encoding the polypeptide comprising the mutant GATA3 peptide sequence.

3. 3. The method of claim 1 or 2, wherein the population of immune cells comprises peripheral blood mononuclear cells (PBMCs).

4. The polypeptide comprising the mutant GATA3 peptide sequence has the following sequence: (a) TLQRSSLWCL (SEQ ID NO: 3753), VLPEPHLAL (SEQ ID NO: 3754), HVLPEPHLAL (SEQ ID NO: 3755), ALQPLQPHA (SEQ ID NO: 3756), AIQPVLWTT (SEQ ID NO: 3757), APAIQPVLWTT (SEQ ID NO: 3758), SMLTGPPARV (SEQ ID NO: 3759), MLTGPPARV (SEQ ID NO: 3760), and / or YMFLKAESKI (SEQ ID NO: 3761); and / or (b) MFLKAESKI (SEQ ID NO: 3762) and / or YMFLKAESKI (SEQ ID NO: 3763); and / or (c) VLWTTPPLQH (SEQ ID NO: 3764), YMFLKAESK (SEQ ID NO: 3765), and / or KIMFATLQR (SEQ ID NO: 3766); and / or (d) FATLQRSSL (SEQ ID NO: 3767), EPHLALQPL (SEQ ID NO: 3768), QPVLWTTPPL (SEQ ID NO: 3769), GPPARVPAV (SEQ ID NO: 3770), MFATLQRSSL (SEQ ID NO: 3771), KPKRDGYMF (SEQ ID NO: 3772), and / or KPKRDGYMFL (SEQ ID NO: 3773), and / or (e) IMKPKRDGYM (SEQ ID NO: 3774), MFATLQRSSL (SEQ ID NO: 3775), FLKAESKIMF (SEQ ID NO: 3776), LHFCRSSIM (SEQ ID NO: 3777), EPHLALQPL (SEQ ID NO: 3778), FATLQRSSL (SEQ ID NO: 3779), ESKIMFATL (SEQ ID NO: 3780), FLKAESKIM (SEQ ID NO: 3781), and / or YMFLKAESKI (SEQ ID NO: 3782). The method according to any one of claims 1 to 3, comprising at least one of:

5. The polypeptide comprising the mutant GATA3 peptide sequence has the following sequence: (a) TLQRSSLWCL (SEQ ID NO: 3783), VLPEPHLAL (SEQ ID NO: 3784), HVLPEPHLAL (SEQ ID NO: 3785), ALQPLQPHA (SEQ ID NO: 3786), AIQPVLWTT (SEQ ID NO: 3787), APAIQPVLWTT (SEQ ID NO: 3788), SMLTGPPARV (SEQ ID NO: 3789), MLTGPPARV (SEQ ID NO: 3790), and / or YMFLKAESKI (SEQ ID NO: 3791); and / or (b) MFLKAESKI (SEQ ID NO: 3792) and / or YMFLKAESKI (SEQ ID NO: 3793); and / or (c) VLWTTPPLQH (SEQ ID NO: 3794), YMFLKAESK (SEQ ID NO: 3795), and / or KIMFATLQR (SEQ ID NO: 3796); and / or (d) FATLQRSSL (SEQ ID NO: 3797), EPHLALQPL (SEQ ID NO: 3798), QPVLWTTPPL (SEQ ID NO: 3799), GPPARVPAV (SEQ ID NO: 3800), MFATLQRSSL (SEQ ID NO: 3801), KPKRDGYMF (SEQ ID NO: 3802), and / or KPKRDGYMFL (SEQ ID NO: 3803), and / or (e) IMKPKRDGYM (SEQ ID NO: 3804), MFATLQRSSL (SEQ ID NO: 3805), FLKAESKIMF (SEQ ID NO: 3806), LHFCRSSIM (SEQ ID NO: 3807), EPHLALQPL (SEQ ID NO: 3808), FATLQRSSL (SEQ ID NO: 3809), ESKIMFATL (SEQ ID NO: 3810), FLKAESKIM (SEQ ID NO: 3811), and / or YMFLKAESKI (SEQ ID NO: 3812). The method according to any one of claims 1 to 3, comprising at least two of the following:

6. 4. The method of any one of claims 1 to 3, wherein the polypeptide comprising the mutant GATA3 peptide sequence comprises a sequence selected from the group consisting of ESKIMFATLQRSSL (SEQ ID NO: 3813), KPKRDGYMFLKAESKI (SEQ ID NO: 3814), SMLTGPPARVPAVPFDLH (SEQ ID NO: 3815), EPCSMLTGPPARVPAVPFDLH (SEQ ID NO: 3816), LHFCRSSIMKPKRDGYMFLKAESKI (SEQ ID NO: 3817), GPPARVPAVPFDLHFCRSSIMKPKRD (SEQ ID NO: 3818), and KPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 3819).

7. The method of any one of claims 1 to 6, wherein the polypeptide comprising the mutant GATA3 peptide sequence comprises PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGL (SEQ ID NO: 3), or EPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 4).

8. The method of any one of claims 1 to 7, wherein the polypeptide comprising the mutant GATA3 peptide sequence comprises SEQ ID NO:

2.

9. 1. A composition comprising a population of T cells comprising a T cell receptor (TCR) specific for a peptide:MHC complex, the peptide of the peptide:MHC complex is a variant GATA3 peptide sequence comprising at least 8 consecutive amino acids of PGRPLQTHVLPEPHLALQPLQPHADHAHADAPAIQPVLWTTPPLQHGHRHGLEPCSMLTGPPARVPAVPFDLHFCRSSIMKPKRDGYMFLKAESKIMFATLQRSSLWCLCSNH (SEQ ID NO: 2); The composition comprises a population of immune cells depleted of cells expressing CD25 and CD14.

10. 10. Use of the composition of claim 9 in the manufacture of a medicament for the treatment of cancer in a subject.

11. the cancer is breast cancer; Optionally, the breast cancer is recurrent or metastatic breast cancer; Optionally, the breast cancer is resistant to anti-estrogen therapy, is MSI breast cancer, is metastatic breast cancer, is Her2-negative breast cancer, is Her2-positive breast cancer, is ER-negative breast cancer, is ER-positive breast cancer, is PR-positive breast cancer, is PR-negative breast cancer, or any combination thereof.

12. The use of claim 10, wherein the subject has experienced disease progression after endocrine therapy in combination with a CDK4 / 6 inhibitor; or the subject has not previously received systemic therapy.

13. The use according to any one of claims 10 to 12, wherein the composition further comprises at least one additional therapeutic agent or treatment.

14. 14. The use of claim 13, wherein the at least one additional therapeutic agent or therapy is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, an APC, a polynucleotide, an oncolytic virus, or any combination thereof.

15. 14. The use of claim 13, wherein the at least one additional therapeutic agent is an anti-PD-1 agent, an anti-PD-L1 agent, an anti-CTLA-4 agent, an anti-CD40 agent, letrozole, fulvestrant, a PI3 kinase inhibitor, and / or a CDK4 / 6 inhibitor.

16. 14. The use of claim 13, wherein the at least one additional therapeutic agent is palbociclib, ribociclib, abemaciclib, seliciclib, dinaciclib, milciclib, roniciclib, atubeciclib, bliciclib, ribiciclib, seliciclib, trilaciclib, voruciclib, or any combination thereof.

17. The at least one additional therapeutic agent is selected from the group consisting of palbociclib (PD0332991); abemaciclib (LY2835219); ribociclib (LEE 011); voruciclib (P1446A-05); fascaplysin; alcyaflavin; 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione; 3-aminothioacridone (3-ATA), trans-4-((6-(ethylamino)-2-((1-(phenylmethyl)-1H-indol-5-yl)amino)-4-pyrimidinyl)amino)-cyclohexano (CINK4); 1,4-dimethoxyacridine-9(10H)-thione (NSC 625987); 2-methyl-5-(p-tolylamino)benzo[d]thiazole-4,7-dione (lyubidin); flavopiridol (alvocidib); seliciclib; dinaciclib; milciclib; roniciclib; atubeciclib; bliciclib; ribiciclib; trilaciclib (G1T28); or any combination thereof.

18. The at least one additional therapeutic agent is selected from the group consisting of wortmannin, demethoxyviridin, LY294002, hibiscone C, idelalisib, copanlisib, duvelisib, taselisib, perifosine, buparlisib, duvelisib, alpelisib (BYL719), umbralisib, (TGR1202), copanlisib (BAY80-6946), PX-866, dactolisib, CUDC-907, voxtalisib (SAR245409, X L765), CUDC-907, ME-401, IPI-549, SF1126, RP6530, INK1117, pictilisib (GDC-0941), XL147 (SAR245408), Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477 or AEZS-13.

19. The use according to any one of claims 10 to 18, wherein the HLA alleles expressed by the subject are unknown at the time of treatment with the composition.

20. The composition of claim 9 for the treatment of cancer in a subject.

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