Neoantigens and uses thereof

Abstract

To provide neoantigens and uses thereof.SOLUTION: Disclosure herein relates to immunotherapeutic compositions comprising immunotherapeutic peptides comprising neoepitopes, polynucleotides encoding the immunotherapeutic peptides, antigen presenting cells comprising the immunotherapeutic peptides or polynucleotides, or T cell receptors specific for the neoepitopes. The use of immunotherapeutic compositions is also disclosed. Novel immunotherapeutic agents and uses thereof are disclosed herein based on the finding of neoantigens generated from individual tumor-specific mutations. Thus, the disclosure provides peptides, polynucleotides encoding the peptides and peptide binding agents that may be used for stimulating immune response toward tumor associated antigen or neoepitopes, or for creating immunogenic compositions or cancer vaccines for use in disease treatment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 687,188, filed June 19, 2018, and U.S. Provisional Patent Application No. 62 / 800,735, filed February 4, 2019, which are incorporated by reference herein in their entireties. [Background technology]

[0002] Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapy takes advantage of the fact that cancer cells often have molecules on their surface known as tumor antigens that can be detected by the immune system, 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 typically 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) to recognize and lyse tumor cells. One of the significant barriers to developing curative tumor-specific immunotherapy is the identification and selection of highly specific and restrictive 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 as mutations, and their corresponding proteins are present only in tumors. They also circumvent central immune 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 technical difficulties in identifying them, selecting optimized antigens, and producing neoantigens for use in vaccines or immunogenic compositions. Therefore, there is still a need to develop 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 some aspects, provided herein are compositions comprising at least one polypeptide comprising two or more mutant RAS peptide sequences selected from the group consisting of: KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and / or VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK; and at least one polynucleotide encoding the at least one polypeptide.

[0006] In some embodiments, the composition comprises a mixture of three or more mutant RAS peptide sequences.

[0007] In some aspects, provided herein is at least one polypeptide comprising two or more mutant RAS peptide sequences, each of which comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a mutation at G12 and a mutation at G12, and further comprising three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of the two or more mutant RAS peptide sequences, wherein the three or more amino acid residues enhance processing of the mutant RAS peptide sequence in cells and / or enhance presentation of an epitope of the mutant RAS peptide sequence; or a composition comprising at least one polynucleotide encoding this at least one polypeptide.

[0008] In some embodiments, three or more amino acid residues heterologous to the mutant RAS protein linked to the N- or C-terminus of two or more mutant RAS peptide sequences comprise amino acid sequences of a CMV protein, e.g., pp65, HIV, or MART-1.

[0009] In some embodiments, the three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of the two or more mutant RAS peptide sequences comprise 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.

[0010] In some embodiments, the three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of the two or more mutant RAS peptide sequences comprise 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.

[0011] In some embodiments, the formula (Xaa N ) N -(Xaa RAS ) P -(Xaa C ) C wherein P is an integer greater than 7; (XaaRAS) P is a mutant RAS peptide sequence comprising at least 8 consecutive amino acids of a mutant RAS protein; and wherein the at least 8 consecutive amino acids are It contains at least 8 consecutive amino acids selected from the group consisting of Gly11 Lys12 Ser13 Ala14 Leu15, and N is (i) 0 or (ii) an integer greater than 2; N ) N is any amino acid sequence heterologous to the mutant RAS protein; C is (i) 0, or (ii) an integer greater than 2; (Xaa C ) C is any amino acid sequence heterologous to the mutant RAS protein; Xaa8 is selected from the group consisting of Asp, Val, Cys, Ala, Arg and Ser; the polypeptide is not KLVVVGAVGVGKSALTIQL; and both N and C are not 0; or a composition comprising at least one polypeptide; or at least one polynucleotide encoding this at least one polypeptide.

[0012] In some embodiments, (Xaa) N and / or (XaaC) C comprises the amino acid sequence of a protein of CMV, such as pp65, HIV or MART-1.

[0013] 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.

[0014] 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.

[0015] In some embodiments, N is 0.

[0016] In some embodiments, C is 0.

[0017] In some embodiments, Xaa1-Xaa2-Val3-Val4-Val5-Gly6-Ala7-Xaa8-Gly9-Xaa 10 wherein Xaa1 is not Ala; with the proviso that if Xaa1 is not Lys, then Xaa2 is Leu, and / or Xaa 10 is Gly; and Xaa2 is not Glu; provided that if Xaa2 is not Leu, then Xaa1 is Lys and / or Xaa 10is Gly; Xaa8 is selected from the group consisting of Asp, Val, Cys, Ala, Arg, and Ser; with the proviso that when Xaa8 is Glu, Xaa1 is not Tyr and / or Xaa2 is not Leu, with the proviso that when Xaa8 is Val, Xaa1 is not Lys; Xaa 10 is any amino acid; provided that Xaa 10is not Gly, then Xaa1 is Lys and / or Xaa2 is Leu; and the polypeptide binds to an HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, and / or HLA-A74:01 molecule. , HLA-A11:01-restricted T cell epitope, HLA-A03:02-restricted T cell epitope, HLA-A30:01-restricted T cell epitope, HLA-A31:01-restricted T cell epitope, HLA-A33:01-restricted T cell epitope, HLA-A33:03-restricted T cell epitope, HLA-A68:01-restricted T cell epitope or HLA-A74:01-restricted T cell epitope; HLA-A02:01-restricted cytotoxic T cell response, HLA-A02:01 Restricted cytotoxic T cell response, HLA-A03:01 restricted cytotoxic T cell response, HLA-A11:01 restricted cytotoxic T cell response, HLA-A03:02 restricted cytotoxic T cell response, HLA-A30:01 restricted cytotoxic T cell response. Answer: HLA-A31:01-restricted cytotoxic T-cell response, HLA-A33:01-restricted cytotoxic T-cell response, HLA-A33:03-restricted cytotoxic T-cell response, HLA-A68:01-restricted cytotoxic T-cell response or HLA-A74: Provided herein are compositions comprising at least one polypeptide that induces a HLA-A02:01-restricted cytotoxic T cell response; and that binds to HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, and / or HLA-A74:01; or at least one polynucleotide encoding said at least one polypeptide.

[0018] In some embodiments, at least one polypeptide comprising one or more mutant RAS peptide sequences, each comprising at least 8 consecutive amino acids of a mutant RAS protein comprising a G12A, G12C, G12D, G12R, G12S, or G12V mutation, and a G12A, G12C, G12D, G12R, G12S, or G12V mutation, and further comprising a peptide comprising a mutation not encoded by the genome of the cancer cell, and having an affinity or predicted affinity for an HLA-A02:01 allele of 150 nM or less and / or a half-life of 2 hours or longer, or a half-life of 2 hours or longer. Provided herein are compositions comprising at least one polypeptide having a short half-life and an affinity or predicted affinity of 150 nM or less for the HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and / or HLA-C08:02 allele; or at least one polynucleotide encoding the at least one polypeptide.

[0019] In some embodiments, the composition further comprises (i) a peptide comprising a peptide sequence in any one of Tables 3-14, or (ii) a polynucleotide encoding a peptide comprising a sequence in Tables 3-14. In some cases, (a) DTAGHEEY、TAGHEEYSAM、DILDTAGHE、DILDTAGH、ILDTAGHEE、ILDTAGHE、DILDTAGHEEY、DTAGHEEYS、LLDILDTAGH、DILDTAGRE、DILDTAGR、ILD TAGREE、ILDTAGRE、CLLDILDTAGR、TAGREEYSAM、REEYSAMRD、DTAGKEEYSAM、CLLDILDTAGK、DTAGKEEY、LLDILDTAGK、ILDTAGKE、ILDTAGKEE、DTAGLEEY、ILDT AGLE、DILDTAGL、ILDTAGLEE、GLEEYSAMRDQY、LLDILDTAGLE、LDILDTAGL、DILDTAGLE、DILDTAGLEEY、AGVGKSAL、GAAGVGKSAL、AAGVGKSAL、CGVGKSAL、ACGV GKSAL、DGVGKSAL、ADGVGKSAL、DGVGKSALTI、GARGVGKSA、KLVVVGARGV、VVVGARGV、SGVGKSAL、VVVGASGVGK、GASGVGKSAL、VGVGKSAL、VVVGAGCVGK、KLVVVGAG C、GDVGKSAL、DVGKSALTI、VVVGAGDVGK、TAGKEEYSAM、DTAGHEEYSAM、TAGHEEYSA、DTAGREEYSAM、TAGKEEYSA、AAGVGKSA、AGCVGKSAL、AGDVGKSAL、AGKEEYSA MR、AGVGKSALTI、ARGVGKSAL、ASGVGKSA、ASGVGKSAL、AVGVGKSA、CVGKSALTI、DILDTAGK、DILDTAGREEY、DTAGHEEYSAMR、DTAGKEEYS、DTAGKEEYSAMR、DTAGLE EYS、DTAGLEEYSA、DTAGLEEYSAMR、DTAGREEYS、DTAGREEYSAMR、GAAGVGKSA、GACGVGKSA、GACGVGKSAL、GADGVGKS、GADGVGKSA、GADGVGKSAL、GAGSVGKSA、GC VGKSAL、GCVGKSALTI、GHEEYSAM、GKEEYSAM、GLEEYSAMR、GREEYSAM、GREEYSAMR、HEEYSAMRD、KEEYSAMRD、KLVVVGASG、LDILDTAGR、LEEYSAMRD、LVVVGARGV、Provided herein are compositions comprising: (a) at least one polypeptide comprising one or more mutant RAS peptide sequences selected from the group consisting of LVVVGASGV, REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK, VGASGVGKSA, VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD, YKLVVVGAR, and DILDTAGKE; or (b) at least one polynucleotide encoding the at least one polypeptide.

[0020] In some embodiments, the composition further comprises (i) a peptide comprising a peptide sequence in any one of Tables 1-14, or (ii) a polynucleotide encoding a peptide comprising a sequence in Tables 1-14.

[0021] In some embodiments, at least one of the mutant RAS peptide sequences comprises an N- or C-terminal amino acid sequence extension of 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, wherein the N- or C-terminal extension is a wild-type RAS amino acid sequence or a non-heterologous RAS amino acid sequence.

[0022] In some embodiments, at least one polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 mutant RAS peptide sequences.

[0023] In some embodiments, the at least one polypeptide comprises at least two polypeptides, or the at least one polynucleotide comprises at least two polynucleotides.

[0024] In some embodiments, at least one of the mutant RAS 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 RAS protein.

[0025] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutant RAS 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 RAS protein.

[0026] In some embodiments, each of the mutant RAS peptide sequences, or each of the two or more RAS 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 RAS protein.

[0027] In some embodiments, at least one polypeptide comprises at least one mutant RAS peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, and / or an HLA-C08:02 allele.

[0028] In some embodiments, at least one polypeptide is selected from the group consisting of HLA-A02:01 and HLA-A03:01 alleles, HLA-A11:01 alleles, HLA-A03:02 alleles, HLA-A30:01 alleles, HLA-A31:01 alleles, HLA-A33:01 alleles, HLA-A33:03 alleles, HLA-A68:01 alleles, and HLA-A74:01 alleles. genes; HLA-A02:01 allele and HLA-C08:02 allele; HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and HLA -C08:02 allele; or HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and allele and HLA-A03:01 allele , HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele.

[0029] In some embodiments, the mutant RAS peptide sequence is a first mutant RAS peptide sequence that binds or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, and / or an HLA-C08:02 allele; and and a second RAS peptide sequence that binds or is predicted to bind to a protein encoded by an HLA allele different from the HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, HLA-A74:01 allele, and / or HLA-C08:02 allele, and wherein the first mutant RAS peptide sequence binds or is predicted to bind to a protein encoded by an HLA allele different from the second mutant RAS peptide sequence.

[0030] In some embodiments, at least one polypeptide comprises at least one mutant RAS 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.

[0031] In some embodiments, at least one polypeptide comprises at least one mutant RAS peptide sequence that binds to a protein encoded by an HLA allele with a stability of 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.

[0032] In some embodiments, the HLA allele is selected from the group consisting of an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele and / or an HLA-C08:02 allele, and any combination thereof.

[0033] In some embodiments, at least one polypeptide comprises at least one of the following sequences: LVVVGACGV, KLVVVGACGV, LVVVGADGV, KLVVVGADGV, LVVVGAVGV, KLVVVGAVGV, VVGACGVGK, VVVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, VVVGAVGVGK, VVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, and VVVGAVGVGK.

[0034] In some embodiments, the mutant RAS peptide sequence comprises at least one or two of the following sequences: KLVVVGACGV, FLVVVGACGL, FMVVVGACGI, FLVVVGACGI, FMVVVGACGV, FLVVVGACGV, MLVVVGACGV, FMVVVGACGL, YLVVVGACGV, KMVVVGACGV, YMVVVGACGV, and MMVVVGACGV.

[0035] In some embodiments, the mutant RAS peptide sequence includes at least one or two of the following sequences: TEYKLVVVGAVGV; WQAGILARKLVVVGAVGVQGQNLKYQ; HSYTTAEKLVVVGAVGVILGVLLLI; PLTEEKIKKLVVVGAVGVEKEGKISK; GALHFKPGSRKLVVVGAVGVAASDFIFLVT; RRANKDATAEKLVVVGAVGVKELKQVASPF; KAFISHEEKRKLVVVGAVGVKKKLINEKKE; TDLSSRFSKSKLVVVGAVGVKKCDISLQFF; FDLGGGTFDVKLVVVGAVGVKSTAGDTHLG; or CLLLHYSVSKKLVVVGAVGVATFYVAVTVP.

[0036] In some embodiments, (Xaa)N comprises the amino acid sequence of IDIIMKIRNA, FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC, FFFFFFFFFFFFFFFFFFFFFFFFFFAAFWFW, IFFIFFIIFFFFFFFFFFFFFFIIIIIIIWEC, FIFFFIIFFFFFFFFFFFIFIFIIIFWEC, TEY, WQAGILAR, HSYTTAE, PLTEEKIK, GALHFKPGSR, RRANKDATAE, KAFISHEEKR, TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK, or MTEYKLVVV.

[0037] In some embodiments, (XaaC)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.

[0038] In some embodiments, the first mutant RAS peptide sequence comprises a first neoepitope of a mutant RAS protein, and the second mutant RAS peptide sequence comprises a second neoepitope of the mutant RAS protein, wherein the first mutant RAS peptide sequence is different from the mutant RAS peptide sequence, the first neoepitope comprises at least one mutant amino acid, and the second neoepitope comprises the same mutant amino acid.

[0039] In some embodiments, at least one of the mutant RAS peptide sequences comprises a mutated amino acid that is not encoded by the genome of the subject's cancer cells.

[0040] In some embodiments, each of the mutant RAS 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.

[0041] In some embodiments, each of the mutant RAS 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.

[0042] In some embodiments, each of the mutant RAS 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.

[0043] In some embodiments, the composition further comprises a different mutant RAS peptide sequence having a G13A, G13C, G13D, G13R, G13S, G13V, G12A, G12C, G12D, G12R, G12S, G12V or Q61 mutation.

[0044] In some embodiments, the composition further comprises an immunomodulatory agent or adjuvant.

[0045] In some embodiments, the adjuvant is polyICLC.

[0046] In some aspects, provided herein is a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable excipient.

[0047] In some embodiments, the pharmaceutical composition comprises a pH modifying agent present at a concentration less than 1 mM or greater than 1 mM.

[0048] In some embodiments, the pharmaceutical composition is a vaccine composition.

[0049] In some embodiments, the pharmaceutical composition is aqueous.

[0050] In some embodiments, one or more of the at least one polypeptide are bounded 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.

[0051] In some embodiments, the pH modifier is a base.

[0052] In some embodiments, the pH modifier is the conjugate base of a weak acid.

[0053] In some embodiments, the pH modifier is a pharmaceutically acceptable salt.

[0054] In some embodiments, the pH modifier is a dicarboxylate or tricarboxylate.

[0055] In some embodiments, the pH modifier is citric acid and / or citrate.

[0056] In some embodiments, the citrate salt is disodium citrate and / or trisodium citrate.

[0057] In some embodiments, the pH modifier is succinic acid and / or succinate.

[0058] In some embodiments, the succinate salt is disodium succinate and / or monosodium succinate.

[0059] In some embodiments, the succinate salt is disodium succinate hexahydrate.

[0060] In some embodiments, the pH modifier is present at a concentration of 0.1 mM to 1 mM.

[0061] In some embodiments, the pharmaceutically acceptable carrier comprises a liquid.

[0062] In some embodiments, the pharmaceutically acceptable carrier comprises water.

[0063] In some embodiments, the pharmaceutically acceptable carrier comprises a sugar.

[0064] In some embodiments, the sugar comprises dextrose or mannitol.

[0065] In some embodiments, dextrose is present at a concentration of 1-10% w / v.

[0066] In some embodiments, the sugar comprises trehalose.

[0067] In some embodiments, the sugar comprises sucrose.

[0068] In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO).

[0069] In some embodiments, DMSO is present at a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%.

[0070] In some embodiments, the pharmaceutically acceptable carrier does not include dimethyl sulfoxide (DMSO).

[0071] In some embodiments, the pharmaceutical composition can be lyophilized.

[0072] In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant.

[0073] In some embodiments, the immunomodulator or adjuvant is poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM-CSF, FLT-3L, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, 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.

[0074] In some embodiments, the immunomodulator or adjuvant comprises poly-ICLC.

[0075] In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is 2:1 to 1:10 v:v.

[0076] 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.

[0077] In some embodiments, the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:3 v:v.

[0078] In some aspects, provided herein is a method of treating a subject having cancer, the method comprising administering to the subject a pharmaceutical composition described herein.

[0079]

[0013] In some aspects, provided herein is a method of treating a subject having cancer, the method comprising administering to the subject a peptide having a sequence of VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK, wherein the subject expresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-C08:02 allele in the subject's genome.

[0080] In some aspects, provided herein are methods of treating a subject having cancer, the method comprising administering to the subject a mutant RAS peptide or a nucleic acid encoding the mutant RAS peptide, wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a mutation at G12, wherein the peptide comprises a mutation at G12 and binds to HLA-A11:01 or HLA-A03:01, and wherein the subject is identified as expressing a protein encoded by an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele.

[0081]

[0013] In some aspects, provided herein are methods of treating a subject having cancer, the methods comprising administering to the subject a peptide comprising the sequence GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, or GAVGVGKSA, wherein the subject expresses a protein encoded by an HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, HLA-A74:01 allele, or HLA-C08:02 allele in the subject's genome that binds to the peptide.

[0082] In some aspects, provided herein are methods of treating a subject having cancer, comprising administering to the subject a first and a second peptide or a nucleic acid encoding the first and second peptides, wherein the first and second peptides comprise at least two of: (1) KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; (2) GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and (3) VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK; and the subject's HLA allele expression is unknown at the time of administration.

[0083] In some aspects, provided herein are methods of treating a subject having cancer, the method comprising administering to the subject a mutant RAS peptide or a nucleic acid encoding the mutant RAS peptide, wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a G12C mutation, the peptide comprises a G12C mutation, and further wherein the peptide comprises a stabilizing mutation not encoded by the genome of the cancer cell, and wherein the subject expresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-C08:02 allele.

[0084] In some aspects, 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-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele, wherein the therapeutic agent is a mutant RAS peptide or a nucleic acid encoding a mutant RAS peptide. Provided herein are methods, wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a mutation at G12, and wherein the peptide comprises a mutation at G12 and binds to a protein encoded by an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele.

[0085] In some embodiments, the method further comprises administering a therapeutic agent to the subject.

[0086] In some aspects, provided herein are methods of treating a subject having cancer, 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 1-14; and (b) administering to the subject: (i) a first mutant RAS peptide that is a peptide for the first HLA allele provided in any one of Tables 1-14, or (ii) a polynucleic acid encoding the first mutant RAS peptide.

[0087] In some embodiments, the method further includes identifying a second protein expressed by the subject, wherein the second protein is encoded by a second HLA allele of the subject, and wherein the second HLA allele is an HLA allele provided in any one of Tables 1-14.

[0088] In some embodiments, the method further comprises administering to the subject (i) a second mutant RAS peptide, wherein the second mutant RAS peptide is a peptide for a second HLA allele provided in any one of Tables 1-14, or (ii) a polynucleic acid encoding the second mutant RAS peptide.

[0089] In some embodiments, the first HLA allele is different from the second HLA allele.

[0090] In some embodiments, the first mutant RAS peptide is different from the second mutant RAS peptide.

[0091] For example, in some embodiments, the first protein expressed by the subject is encoded by HLA-A03:01, e.g., as provided in Table 11, and the method includes administering to the subject a first mutant RAS peptide or a polynucleic acid encoding the first mutant RAS peptide, the first mutant RAS peptide comprising a sequence of VVGASGVGK.

[0092] For example, in some embodiments, the first protein expressed by the subject is encoded by HLA-A03:01, e.g., as provided in Table 5, and the method includes administering to the subject a first mutant RAS peptide or a polynucleic acid encoding the first mutant RAS peptide, the first mutant RAS peptide comprising a sequence of CLLDILDTAGK.

[0093] As another example, in some embodiments, the second protein expressed by the subject is encoded by HLA-A11:01, e.g., as provided in Table 11, and the method includes administering to the subject a second mutant RAS peptide or a polynucleic acid encoding the second mutant RAS peptide, the second mutant RAS peptide comprising a sequence of VVVGASGVGK.

[0094] As yet another example, in some embodiments, the second protein expressed by the subject is encoded by HLA-C08:02, e.g., as provided in Table 9, and the method includes administering to the subject a second mutant RAS peptide or a polynucleic acid encoding the second mutant RAS peptide, the second mutant RAS peptide comprising a sequence of GADGVGKSAL.

[0095] In some embodiments, an immune response is raised in the subject.

[0096] In some embodiments, the immune response is a humoral response.

[0097] In some embodiments, the mutant RAS peptide sequences are administered simultaneously, separately or sequentially.

[0098] In some embodiments, the first peptide is administered sequentially after a period sufficient for the second peptide to activate the second T cells.

[0099] In some embodiments, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer, and liver cancer.

[0100] In some embodiments, the method further comprises administering at least one additional therapeutic agent or modality.

[0101] In some embodiments, the at least one additional therapeutic agent or modality 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.

[0102] 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.

[0103] In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after the step of administering the mutant RAS peptide sequence.

[0104] 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) (i) KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; (ii) GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and / or (iii)VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK or a pharmaceutically acceptable salt thereof; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 2) 2. The composition of claim 1, comprising a mixture of three or more of said mutant RAS peptide sequences. (Item 3) (a) at least one polypeptide or a pharmaceutically acceptable salt thereof comprising two or more mutant RAS peptide sequences, each of said mutant RAS peptide sequences being: (i) at least 8 consecutive amino acids of a mutant RAS protein containing a mutation at G12; and (ii) the mutation in G12 Including, a polypeptide or a pharmaceutically acceptable salt thereof, further comprising three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of two or more of the mutant RAS peptide sequences, wherein the three or more amino acid residues enhance processing of the mutant RAS peptide sequence and / or enhance presentation of an epitope of the mutant RAS peptide sequence in a cell; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 4) 4. The composition of claim 3, wherein the three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of the two or more mutant RAS peptide sequences comprise amino acid sequences of a CMV protein, e.g., pp65, HIV, or MART-1. (Item 5) 5. The composition of claim 3 or 4, wherein the three or more amino acid residues heterologous to the mutant RAS protein linked to the N-terminus or C-terminus of the two or more mutant RAS peptide sequences comprise 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. (Item 6) 6. The composition of any one of items 3 to 5, wherein the three or more amino acid residues heterologous to the mutant RAS protein linked to the N- or C-terminus of the two or more mutant RAS peptide sequences comprise 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. (Item 7) Formula (a) (Xaa N ) N -(Xaa RAS ) P -(Xaa C ) C or a pharmaceutically acceptable salt thereof, During the ceremony, P is an integer greater than 7; (Xaa RAS ) P is a mutant RAS peptide sequence comprising at least 8 consecutive amino acids of a mutant RAS protein; and said at least 8 consecutive amino acids are Lys1Leu2Val3Val4Val5Gly6Ala7Xaa8Gly9Val 10 Gly 11 Lys 12 Ser 13 Ala 14 Leu 15 and at least 8 consecutive amino acids of N is (i) 0, or (ii) an integer greater than 2; (Xaa N ) N is any amino acid sequence heterologous to said mutant RAS protein; C is (i) 0, or (ii) an integer greater than 2; (Xaa C )C is any amino acid sequence heterologous to said mutant RAS protein; Xaa8 is selected from the group consisting of Asp, Val, Cys, Ala, Arg, and Ser; the polypeptide is not KLVVVGAVGVGKSALTIQL; at least one polypeptide or a pharmaceutically acceptable salt thereof, wherein when N is 0, C is not 0 and when C is 0, N is not 0; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 8) (Xaa) N and / or (Xaa C ) C 8. The composition of claim 7, wherein the amino acid sequence of the protein is a CMV protein, such as pp65, HIV, or MART-1. (Item 9) 9. The composition according to item 7 or 8, wherein N and / or C is an integer 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. (Item 10) 10. The composition of any one of items 7 to 9, wherein N and / or C is an integer 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. (Item 11) 11. The composition of any one of items 7 to 10, wherein N is 0. (Item 12) 11. The composition of any one of items 7 to 10, wherein C is 0. (Item 13) (a)Xaa1-Xaa2-Val3-Val4-Val5-Gly6-Ala7-Xaa8-Gly9-Xaa 10 or a pharmaceutically acceptable salt thereof, During the ceremony, Xaa1 instead of Ala; provided that if Xaa1 is not Lys, then Xaa2 is Leu and / or Xaa 10 is Gly; Xaa2 instead of Glu; provided that if Xaa2 is not Leu, then Xaa1 is Lys and / or Xaa 10 is Gly; Xaa8 is selected from the group consisting of Asp, Val, Cys, Ala, Arg, and Ser; with the proviso that when Xaa8 is Glu, Xaa1 is not Tyr and / or Xaa2 is not Leu, However, if Xaa8 is Val, then Xaa1 is not Lys; Xaa 10 is any amino acid; However, Xaa 10 is not Gly, then Xaa1 is Lys and / or Xaa2 is Leu; The polypeptide is an HLA-A02:01-restricted T cell epitope, an HLA-A03 ... A-A11:01-restricted T cell epitope, HLA-A03:02-restricted T cell epitope, HLA-A30:01-restricted T cell epitope, HLA-A31:01-restricted T cell epitope, HLA-A33:01-restricted T cell epitope, HLA-A33:03-restricted T cell epitope, HLA-A68:01-restricted T cell epitope, or HLA-A74:01-restricted T cell epitope, HLA-A02:01-restricted cytotoxic T-cell response, HLA-A02:01-restricted cytotoxic T-cell response, HLA-A03:01-restricted cytotoxic T-cell response, HLA-A11:01-restricted cytotoxic T-cell response, HLA-A03:02-restricted cytotoxic T-cell response, HLA-A30:01-restricted cell inducing a toxic T-cell response, an HLA-A31:01-restricted cytotoxic T-cell response, an HLA-A33:01-restricted cytotoxic T-cell response, an HLA-A33:03-restricted cytotoxic T-cell response, an HLA-A68:01-restricted cytotoxic T-cell response, or an HLA-A74:01-restricted cytotoxic T-cell response; at least one polypeptide or a pharmaceutically acceptable salt thereof that binds to HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A03:02, HLA-A30:01, HLA-A31:01, HLA-A33:01, HLA-A33:03, HLA-A68:01, and / or HLA-A74:01; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 14) (a) at least one polypeptide or a pharmaceutically acceptable salt thereof comprising one or more mutant RAS peptide sequences, each of said mutant RAS peptide sequences comprising: (i) at least 8 consecutive amino acids of a mutant RAS protein containing a G12A, G12C, G12D, G12R, G12S, or G12V mutation; and (ii) comprises the G12A, G12C, G12D, G12R, G12S, or G12V mutation; Furthermore, the peptide (i) contains a mutation not encoded by the genome of the cancer cell and has an affinity or predicted affinity for the HLA-A02:01 allele of 150 nM or less and / or a half-life of 2 hours or longer; or (j) at least one polypeptide or a pharmaceutically acceptable salt thereof having a half-life of 2 hours or longer and an affinity or predicted affinity of 150 nM or less for the HLA-A02:01 allele, HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele and / or HLA-C08:02 allele; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 15) 15. The composition of any one of items 1 to 14, further comprising (i) a peptide comprising a peptide sequence in any one of Tables 3 to 14, or (ii) a polynucleotide encoding said peptide comprising a sequence in Tables 3 to 14. (Item 16) (a) DTAGHEEY、TAGHEYSAM、DILDTAGHE、DILDTAGH、ILDTAGHEE、ILDTAGHE、DILDTAGHEY、DTAGHEEYS、LLDILDTAGH、DILDTAGRE、DILDTAGR、ILDTAGREE、I LDTAGRE、CLLDILDTAGR、TAGREEYSAM、REYSAMRD、DTAGKEEYSAM、CLLDILDTAGK、DTAGKEY、LLDILDTAGK、ILDTAGKE、ILDTAGKEE、DTAGLEEY、ILDTAGLE、DIL DTAGL、ILDTAGLEE、GLEEYSAMRDQY、LLDILDTAGLE、LDILDTAGL、DILDTAGLE、DILDTAGLEEY、AGVGKSAL、GAAGVGKSAL、AAGVGKSAL、CGVGKSAL、ACGVGKSAL、DGV GKSAL、ADGVGKSAL、DGVGKSALTI、GARGVGKSA、KLVVVGARGV、VVVGARGV、SGVGKSAL、VVVGASGVGK、GASGVGKSAL、VGVGKSAL、VVVGAGCVGK、KLVVVGAGC、GDVGKSA L. ALTI、ARGVGKSAL、ASGVGKSA、ASGVGKSAL、AVGVGKSA、CVGKSALTI、DILDTAGK、DILDTAGREEY、DTAGHEEYSAMR、DTAGKEEYS、DTAGKEEYSAMR、DTAGLEEYS、DTAGLEYS EEYSA、DTAGLEEYSAMR、DTAGREEYS、DTAGREEYSAMR、GAAGVGKSA、GACGVGKSA、GACGVGKSAL、GADGVGKS、GAGDVGKSA、GAGDVGKSAL、GASGVGKSA、GCVGKSAL、GCVGKSALTI、GHEEYSAM、GKEEYSAM、GLEEYSAMR、GREEYSAM、GREEYSAMR、HEEYSAMRD、KEEYSAMRD、KLVVVGASG、LDILDTAGR、LEEYSAMRD、LVVVGARGV、LVVVGASGV、At least one polypeptide comprising one or more mutant RAS peptide sequences selected from the group consisting of REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK, VGASGVGKSA, VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD, YKLVVVGAR, and DILDTAGKE, or a pharmaceutically acceptable salt thereof; or (b) at least one polynucleotide encoding said at least one polypeptide; A composition comprising: (Item 17) (i) a peptide comprising a peptide sequence in any one of Tables 1 to 14, or (ii) a polynucleotide encoding the peptide comprising a sequence in Tables 1 to 14 17. The composition of claim 16, further comprising: (Item 18) 18. The composition of any one of items 1 to 17, wherein at least one of the mutant RAS peptide sequences comprises an N- or C-terminal amino acid sequence extension of 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, and wherein the N- or C-terminal extension is a wild-type RAS amino acid sequence or a non-heterologous RAS amino acid sequence. (Item 19) 19. The composition of any one of items 1 to 18, wherein the at least one polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9 or 10 mutant RAS peptide sequences. (Item 20) 20. The composition of any one of items 1 to 19, wherein the at least one polypeptide comprises at least two polypeptides or the at least one polynucleotide comprises at least two polynucleotides. (Item 21) 21. The composition of any one of items 1 to 20, wherein at least one of the mutant RAS 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 RAS protein. (Item 22) 22. The composition of any one of items 1 to 21, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the mutant RAS 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 mutant RAS protein. (Item 23) 23. The composition of any one of items 1 to 22, wherein each of the mutant RAS peptide sequences or each of two or more of the RAS 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 RAS protein. (Item 24) 24. The composition of any one of items 1 to 23, wherein the at least one polypeptide comprises at least one mutant RAS peptide sequence that binds or is predicted to bind to a protein encoded by the HLA-A02:01 allele, the HLA-A03:01 allele, the HLA-A11:01 allele and / or the HLA-C08:02 allele. (Item 25) the at least one polypeptide (a) HLA-A02:01 allele and HLA-A03:01 allele, HLA-A11:01 allele, HLA-A03:02 allele, HLA-A30:01 allele, HLA-A31:01 allele, HLA-A33:01 allele, HLA-A33:03 allele, HLA-A68:01 allele, or HLA-A74:01 allele; (b) HLA-A02:01 allele and HLA-C08:02 allele; (c) the HLA-A03:01 allele, the HLA-A11:01 allele, the HLA-A03:02 allele, the HLA-A30:01 allele, the HLA-A31:01 allele, the HLA-A33:01 allele, the HLA-A33:03 allele, the HLA-A68:01 allele, or the HLA-A74:01 allele and the HLA-C08:02 allele; or (d) an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele and an allele and an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele 25. The composition of any one of items 1 to 24, comprising at least one mutant RAS peptide sequence that binds to or is predicted to bind to a protein encoded by (Item 26) the mutant RAS peptide sequence is (a) a first variant RAS peptide sequence that binds to or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, and / or an HLA-C08:02 allele; and (b) a second RAS peptide sequence that binds to or is predicted to bind to a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, and / or an HLA-C08:02 allele. Including, 26. The composition of any one of items 1 to 25, wherein the first mutant RAS peptide sequence binds or is predicted to bind to a protein encoded by a different HLA allele than the second mutant RAS peptide sequence. (Item 27) 27. The composition of any one of items 1 to 26, wherein the at least one polypeptide comprises at least one mutant RAS 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. (Item 28) 28. The composition of any one of items 1 to 27, wherein the at least one polypeptide comprises at least one mutant RAS peptide sequence that binds to a protein encoded by an HLA allele with a stability of 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. (Item 29) 29. The composition of item 27 or 28, wherein the HLA allele is selected from the group consisting of an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele and / or an HLA-C08:02 allele and any combination thereof. (Item 30) 30. The composition of any one of items 1 to 29, wherein the at least one polypeptide comprises at least one of the following sequences: LVVVGACGV, KLVVVGACGV, LVVVGADGV, KLVVVGADGV, LVVVGAVGV, KLVVVGAVGV, VVGACGVGK, VVVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, VVVGAVGVGK, VVGACGVGK, VVGADGVGK, VVVGADGVGK, VVGAVGVGK, and VVVGAVGVGK. (Item 31) 31. The composition of any one of items 1 to 30, wherein the mutant RAS peptide sequence comprises at least one or two of the following sequences: KLVVVGACGV, FLVVVGACGL, FMVVVGACGI, FLVVVGACGI, FMVVVGACGV, FLVVVGACGV, MLVVVGACGV, FMVVVGACGL, YLVVVGACGV, KMVVVGACGV, YMVVVGACGV, and MMVVVGACGV. (Item 32) The mutant RAS peptide sequence has the following sequence: (a)TEYKLVVVGAVGV; (b)WQAGILARKLVVVGAVGVQGQNLKYQ; (c)HSYTTAEKLVVVGAVGVILGVLLLI; (d)PLTEEKIKKLVVVGAVGVEKEGKISK; (e)GALHFKPGSRKLVVVGAVGVAASDFIFLVT; (f)RRANKDATAEKLVVVGAVGVKELKQVASPF; (g)KAFISHEEKRKLVVVGAVGVKKKLINEKKE; (h)TDLSSRFSSKSKLVVVGAVGVKKCDISLQFF; (i)FDLGGGTFDVKLVVVGAVGVKSTAGDTHLG; or (j)CLLLHYSVSKLVVVGAVGVATFYVAVTVP 32. The composition according to any one of items 1 to 31, comprising at least one or two of: (Item 33) (Xaa) N 33. The composition of any one of items 1 to 32, wherein the amino acid sequence of IDIIMKIRNA, FFFFFFFFFFFFFFFFFFFFIIFFIFFWMC, FFFFFFFFFFFFFFFFFFFFFFFFFFAAFWFW, IFFIFFIIFFFFFFFFFFFFFFIIIIIIIWEC, FIFFFIIFFFFFFFFFFFIFIFIIIFWEC, TEY, WQAGILAR, HSYTTAE, PLTEEKIK, GALHFKPGSR, RRANKDATAE, KAFISHEEKR, TDLSSRFSKS, FDLGGGTFDV, CLLLHYSVSK, or MTEYKLVVV. (Item 34) (Xaa C ) C34. The composition of any one of items 1 to 33, wherein the amino acid sequence comprises KKNKKDDIKD, AGNDDDDDDDDDDDDDDDDDDDKKDKDDDDDD, AGNKKKKKKKNNNNNNNNNNNNNNNNNNNN, AGRDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD, GKSALTIQL, GKSALTI, QGQNLKYQ, ILGVLLLI, EKEGKISK, AASDFIFLVT, KELKQVASPF, KKKLINEKKE, KKCDISLQFF, KSTAGDTHLG, ATFYVAVTVP, LTIQLIQNHFVDEYDPTIEDSYRKQVVIDG, or TIQLIQNHFVDEYDPTIEDSYRKQVVIDGE. (Item 35) 35. The composition of any one of items 1 to 34, wherein a first mutant RAS peptide sequence comprises a first neoepitope of a mutant RAS protein, and a second mutant RAS peptide sequence comprises a second neoepitope of a mutant RAS protein, wherein the first mutant RAS peptide sequence is different from the mutant RAS peptide sequence, the first neoepitope comprises at least one mutant amino acid, and the second neoepitope comprises the same mutant amino acid. (Item 36) 36. The composition of any one of items 1 to 35, wherein at least one of the mutant RAS peptide sequences comprises a mutant amino acid that is not encoded by the genome of the subject's cancer cell. (Item 37) 37. The composition of any one of items 1 to 36, wherein each of the mutant RAS 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. (Item 38) 37. The composition of any one of items 1 to 36, wherein each of the mutant RAS 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. (Item 39) 37. The composition of any one of items 1 to 36, wherein each of the mutant RAS 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. (Item 40) 40. The composition of any one of items 1 to 39, further comprising a different mutant RAS peptide sequence having a G13A, G13C, G13D, G13R, G13S, G13V, G12A, G12C, G12D, G12R, G12S, G12V or Q61 mutation. (Item 41) 41. The composition of any one of items 1 to 40, further comprising an immunomodulatory agent or adjuvant. (Item 42) 42. The composition of claim 41, wherein the adjuvant is polyICLC. (Item 43) (a) a composition according to any one of items 1 to 42, and (b) a pharmaceutically acceptable excipient 10. A pharmaceutical composition comprising: (Item 44) 44. The pharmaceutical composition of item 43, comprising a pH modifying agent present at a concentration of less than 1 mM or more than 1 mM. (Item 45) 45. The pharmaceutical composition according to item 43 or 44, which is a vaccine composition. (Item 46) 46. ​​The pharmaceutical composition according to any one of items 43 to 45, which is aqueous. (Item 47) one or more of said at least one polypeptide: (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 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 47. The pharmaceutical composition according to any one of items 43 to 46, wherein (Item 48) 48. The pharmaceutical composition according to any one of items 43 to 47, wherein the pH modifier is a base. (Item 49) 49. The pharmaceutical composition according to any one of items 43 to 48, wherein the pH modifier is a conjugate base of a weak acid. (Item 50) 50. The pharmaceutical composition according to any one of items 43 to 49, wherein the pH modifier is a pharmaceutically acceptable salt. (Item 51) 51. The pharmaceutical composition according to any one of items 43 to 50, wherein the pH modifier is a dicarboxylate or tricarboxylate. (Item 52) 51. The pharmaceutical composition according to any one of items 43 to 50, wherein the pH modifier is citric acid and / or citrate salt. (Item 53) 53. The pharmaceutical composition of item 52, wherein the citrate salt is disodium citrate and / or trisodium citrate. (Item 54) 51. The pharmaceutical composition according to any one of items 43 to 50, wherein the pH modifier is succinic acid and / or a succinate salt. (Item 55) 55. The pharmaceutical composition according to item 54, wherein the succinate salt is disodium succinate and / or monosodium succinate. (Item 56) 56. The pharmaceutical composition of claim 55, wherein the succinate salt is disodium succinate hexahydrate. (Item 57) 56. The pharmaceutical composition according to any one of items 43 to 55, wherein the pH modifying agent is present in a concentration of 0.1 mM to 1 mM. (Item 58) 58. The pharmaceutical composition according to any one of items 43 to 57, wherein the pharmaceutically acceptable carrier comprises a liquid. (Item 59) 59. The pharmaceutical composition according to any one of items 43 to 58, wherein the pharmaceutically acceptable carrier comprises water. (Item 60) 60. The pharmaceutical composition according to any one of items 43 to 59, wherein the pharmaceutically acceptable carrier comprises a sugar. (Item 61) 61. The pharmaceutical composition of claim 60, wherein the sugar comprises dextrose. (Item 62) 62. The pharmaceutical composition according to item 61, wherein the dextrose is present in a concentration of 1 to 10% w / v. (Item 63) 63. The pharmaceutical composition of any one of items 60 to 62, wherein the sugar comprises trehalose. (Item 64) Item 65. The pharmaceutical composition according to any one of items 60 to 63, wherein the sugar comprises sucrose. 65. The pharmaceutical composition of any one of items 43 to 64, wherein the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO). (Item 66) 66. The pharmaceutical composition of item 65, wherein the DMSO is present in a concentration of 0.1% to 10%, 0.5% to 5%, or 1% to 3%. (Item 67) 65. The pharmaceutical composition according to any one of items 43 to 64, wherein the pharmaceutically acceptable carrier does not comprise dimethyl sulfoxide (DMSO). (Item 68) 68. The pharmaceutical composition according to any one of items 43 to 67, which is lyophilizable. (Item 69) 69. The pharmaceutical composition according to any one of items 43 to 68, further comprising an immunomodulator or adjuvant. (Item 70) The immunomodulator or adjuvant is selected from the group consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonist, dSLIM, GM-CSF, FLT-3L, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide. 70. The pharmaceutical composition of item 69, selected from the group consisting of 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. (Item 71) 71. The pharmaceutical composition of claim 69 or 70, wherein the immunomodulator or adjuvant comprises poly-ICLC. (Item 72) 72. The pharmaceutical composition according to item 71, wherein the ratio of poly-ICLC to peptide in the pharmaceutical composition is 2:1 to 1:10 v:v. (Item 73) 73. The pharmaceutical composition of claim 72, wherein 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. (Item 74) 74. The pharmaceutical composition of claim 73, wherein the ratio of poly-ICLC to peptide in the pharmaceutical composition is about 1:3 v:v. (Item 75) 75. A method of treating a subject having cancer, comprising administering to the subject a pharmaceutical composition according to any one of items 43 to 74. (Item 76) 1. A method of treating a subject having cancer, comprising the step of administering to the subject a peptide having the sequence VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK, wherein the subject expresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-C08:02 allele in the subject's genome. (Item 77) 1. A method of treating a subject having cancer, comprising: 10. A method comprising administering to the subject a mutant RAS peptide or a nucleic acid encoding the mutant RAS peptide, wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a mutation at G12, wherein the peptide comprises the mutation at G12 and binds to HLA-A11:01 or HLA-A03:01, and wherein the subject is identified as expressing a protein encoded by an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele. (Item 78) 1. A method of treating a subject having cancer, comprising administering to the subject a peptide comprising the sequence GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, or GAVGVGKSA; The method, wherein the subject expresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-C08:02 allele in the subject's genome that binds to the peptide. (Item 79) 1. A method of treating a subject having cancer, comprising administering to the subject a first and a second peptide or a nucleic acid encoding the first and second peptides, wherein the first and second peptides comprise at least two of: (1) KLVVVGADGV, KLVVVGACGV, KLVVVGAVGV, LVVVGADGV, LVVVGACGV, LVVVGAVGV; (2) GADGVGKSAL, GACGVGKSAL, GAVGVGKSAL, GADGVGKSA, GACGVGKSA, GAVGVGKSA; and (3) VVGADGVGK, VVGACGVGK, VVGAVGVGK, VVVGADGVGK, VVVGACGVGK, VVVGAVGVGK; and the subject's HLA allele expression is unknown at the time of administration. (Item 80) 81. A method for treating a subject having cancer, comprising administering to the subject a mutant RAS peptide or a nucleic acid encoding the mutant RAS peptide, wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a G12C mutation, the peptide comprising the G12C mutation, and further comprising a stabilizing mutation not encoded by the genome of a cancer cell, wherein the subject expresses a protein encoded by an HLA-A02:01 allele, an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, an HLA-A74:01 allele, or an HLA-C08:02 allele. 1. A method of identifying a subject having cancer as a candidate for a therapeutic agent, the method comprising the step of identifying the subject as expressing a protein encoded by an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele, wherein the therapeutic agent is a mutant RAS peptide or a nucleic acid encoding the mutant RAS peptide. wherein the mutant RAS peptide comprises at least 8 consecutive amino acids of a mutant RAS protein comprising a mutation at G12, and wherein the peptide comprises the mutation at G12 and binds to a protein encoded by an HLA-A03:01 allele, an HLA-A11:01 allele, an HLA-A03:02 allele, an HLA-A30:01 allele, an HLA-A31:01 allele, an HLA-A33:01 allele, an HLA-A33:03 allele, an HLA-A68:01 allele, or an HLA-A74:01 allele. (Item 82) 82. The method of claim 81, further comprising administering the therapeutic agent to the subject. (Item 83) 1. A method of treating a subject having cancer, comprising: (a) identifying a first protein expressed by the subject, wherein the first protein is encoded by a first HLA allele of the subject, wherein the first HLA allele is an HLA allele provided in any one of Tables 1-14; and (b) administering to the subject (i) a first mutant RAS peptide that is a peptide for the first HLA allele provided in any one of Tables 1-14, or (ii) a polynucleic acid encoding the first mutant RAS peptide. A method comprising: (Item 84) 84. The method of claim 83, further comprising identifying a second protein expressed by the subject, wherein the second protein is encoded by a second HLA allele of the subject, and the second HLA allele is an HLA allele provided in any one of Tables 1-14. (Item 85) 85. The method of item 84, further comprising administering to the subject (i) a second mutant RAS peptide, wherein the second mutant RAS peptide is a peptide for the second HLA allele provided in any one of Tables 1 to 14, or (ii) a polynucleic acid encoding the second mutant RAS peptide. (Item 86) 86. The method of item 84 or 85, wherein the first HLA allele is different from the second HLA allele. (Item 87) 86. The method of claim 84 or 85, wherein the first mutant RAS peptide is different from the second mutant RAS peptide. (Item 88) 88. The method of any one of items 75 to 87, wherein an immune response is raised in the subject. (Item 89) 89. The method of item 88, wherein the immune response is a humoral response. (Item 90) 90. The method of any one of items 75 to 89, wherein the mutant RAS peptide sequences are administered simultaneously, separately or sequentially. (Item 91) 91. The method of claim 90, wherein the first peptide is administered sequentially after a period sufficient for the second peptide to activate second T cells. (Item 92) 92. The method of any one of items 75 to 91, wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, uterine cancer and liver cancer. (Item 93) 93. The method of any one of items 75 to 92, further comprising administering at least one additional therapeutic agent or modality. (Item 94) 94. The method of item 93, wherein the at least one additional therapeutic agent or modality 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 95) 95. The method of item 94, 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, or an anti-CD40 agent. (Item 96) 96. The method of claim 94 or 95, wherein the additional therapeutic agent is administered before, simultaneously with, or after administering the mutant RAS peptide sequence. [Brief explanation of the drawings]

[0105] [Figure 1] FIG. 1 shows an exemplary workflow for the determination of RAS epitopes capable of inducing CD8+ and / or CD4+ T cells.

[0106] [Figure 2-1]2A and 2B show an overview of an experiment demonstrating that the predicted RAS G12C epitope for HLA-A11:01 (left) and the RAS G12V epitope for HLA-A11:01 (right) can be detected by mass spectrometry. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0107] [Figure 3-1] 3A and 3B show exemplary multimer plots of RAS mutant-specific CD8+ T cell responses. [Figure 3-2] Same as above.

[0108] [Figure 3-3] Figure 3C shows an example of results showing antigen-specific CD8+ T cell responses to a long peptide containing the minimal epitope for KRAS G12V on HLA-A11:01. The sequence of the long peptide used to stimulate the response is shown, as well as the sequence of the minimal epitope used for multimer staining.

[0109] [Figure 4-1] Figure 4A is a graph showing antigen-specific induction of IFNγ. IFNγ levels are shown for samples mock-transduced or transduced with a lentiviral expression vector encoding a mutant RAS peptide.

[0110] [Figure 4-2] Figure 4B is a graph showing upregulation of active caspase 3 on target cells. Shown is the percent viable caspase-A positive target cells for samples mock transduced or transduced with a lentiviral expression vector encoding a mutant RAS peptide.

[0111] [Figure 5-1]Figure 5A is a graph showing antigen-specific induction of IL-2 by co-culturing T cells expressing TCRs specific for mutant RAS peptides with target cells transduced with 9-mer or 11-mer mutant RAS peptides. The data demonstrate that RAS-specific T cells recognize the mutated cells and upregulate cytotoxic machinery.

[0112] [Figure 5-2] Figure 5B is a graph showing antigen-specific induction of IL-2 by co-culturing T cells expressing TCRs specific for 9-mer or 11-mer mutant RAS peptides with target cells loaded with increasing concentrations of mutant RAS peptides. The data indicate that RAS-specific T cells recognize the mutated cells and upregulate cytotoxic machinery.

[0113] [Figure 5-3] Figure 5C is a graph showing antigen-specific induction of IL-2 by co-culturing T cells expressing a TCR specific for a mutant RAS peptide with target cells transduced with a 9-mer mutant RAS peptide. The data indicate that RAS-specific T cells recognize the mutated cells and upregulate cytotoxic machinery.

[0114] [Figure 5-4] Figure 5D is a graph showing antigen-specific induction of IL-2 by co-culturing T cells expressing a TCR specific for a 9-mer mutant RAS peptide with target cells loaded with increasing concentrations of the mutant RAS peptide. The data demonstrate that RAS-specific T cells recognize the mutated cells and upregulate cytotoxic machinery.

[0115] [Figure 5-5]Figures 5E-5H show the antigen-specific cytotoxic activity of T cells expressing TCRs specific for mutant RAS peptides. These data demonstrate that RAS-specific TCRs can elicit specific recognition of cells bearing the mutated peptide and appropriate MHC-I and upregulate cytotoxicity.

[0116] [Figure 6] FIG. 6 shows FACS analysis of antigen-specific induction of IFNγ levels in CD4+ cells from healthy donors stimulated with APCs loaded or not with mutant RAS peptides. DETAILED DESCRIPTION OF THE INVENTION

[0117] Described herein are new 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.

[0118] The following description and examples illustrate embodiments of the present disclosure in detail. 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 recognize that there are numerous variations and modifications of the present disclosure that fall within its scope.

[0119] All terms are intended to be understood as 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 pertains.

[0120] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0121] While various features of the present disclosure may be described in terms of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may for clarity be described herein in terms of separate embodiments, the present disclosure may also be practiced in a single embodiment.

[0122] The following definitions are provided to assist those skilled in the art and are directed to the present application, and are not to be construed as limiting any related or unrelated matter, for example, any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in practice for testing the present disclosure, 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. I. Definition

[0123] The terminology used herein is for the purpose of describing particular instances 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.

[0124] In this application, the use of "or" means "and / or" unless otherwise stated. The terms "and / or" and "any combinations thereof," as well as their grammatical equivalents, may be used interchangeably when used herein. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B and / or C" or "A, B, C, or any combinations 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.

[0125] The term "about" or "approximately" can mean within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or within more than one standard deviation, according to practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of within 5-fold, more preferably within 2-fold, of a value. When particular values ​​are described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0126] As used in the specification and 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 implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0127] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with those embodiments 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.

[0128] The "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in controlling the cellular interactions responsible for the physiological immune response. 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" should be understood to mean a protein that is capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential lymphocyte epitopes (e.g., T-cell epitopes and B-cell epitopes) and transporting them to the cell surface, where they are presented to specific cells, particularly cytotoxic T lymphocytes, T-helper cells, or B-cells. The major histocompatibility complex in the genome comprises gene regions whose gene products, expressed on the cell surface, bind and present endogenous and / or foreign antigens and are therefore important for 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 these different roles.

[0129] "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 thEd., Lange Publishing, Los Altos, Calif. (1994).

[0130] As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to a polymer of amino acid residues, typically L-amino acids, connected to one another by peptide bonds 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, can be in either their neutral (uncharged) or salt form, and can contain or be free of modifications, such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptides described herein. A "mature protein" is a protein that is full-length and optionally contains glycosylation or other modifications typical of that protein 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-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. These include β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-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 the expression of the polypeptides described herein in engineered cells may be associated with 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.

[0131] A peptide or polypeptide can include at least one flanking sequence. The term "flanking sequence" as used herein refers to a fragment or region of a peptide that is not part of the epitope.

[0132] An "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" is one 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 peptides containing allele-specific motifs that induce a B lymphocyte response. Thus, the immunogenic peptides described herein are capable of binding to the appropriate HLA molecule and subsequently inducing a CTL (cytotoxic) or HTL (and humoral) response against the peptide.

[0133] "Neoantigen" refers to a class of tumor antigens that arise from tumor-specific alterations in proteins. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, substitutions in protein sequences, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides.

[0134] 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 a nucleic acid (DNA or RNA) that encodes an amino acid or amino acid mimetic.

[0135] The terms "neoepitope," "tumor-specific neoepitope," or "tumor antigen" refer to an epitope or antigenic determinant region that is absent in, for example, 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 due to one or more mutations in the diseased cells, e.g., cancer cells, the sequence of the epitope is altered to create a neoepitope. The term "neoepitope," as used herein, 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, resulting in specificity of the interaction with HLA. In some cases, the anchor residue may be at a canonical anchor position. In other cases, the anchor residues may be 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. In the peptide-binding groove, specific amino acids form pockets that accommodate the corresponding side chains of the anchor residues of the presented neoepitope. Peptide binding preferences exist among different alleles of both HLA I and HLA II molecules. HLA class I molecules bind short neoepitopes, whose N- and C-termini are anchored in 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 accommodated by their central protrusions, resulting in binding neoepitopes of approximately 8 to 12 amino acids. Neoepitopes that bind to HLA class II proteins are not limited in size and can range from approximately 16 to 25 amino acids. The neoepitope binding groove in HLA class II molecules is open at both ends, allowing 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 adjacent region is also important for peptide specificity for HLA class II alleles. In some cases, the anchor residue adjacent region is the N-terminal residue. In other cases, the anchor residue adjacent region is the C-terminal residue. In yet other cases, the anchor residue adjacent region is both the N-terminal and C-terminal residues. In some cases, the anchor residue adjacent region is flanked by at least two anchor residues. An anchor residue adjacent region flanked by anchor residues is a "separation region."

[0136] " Reference " can be used to correlate and compare the results obtained from tumor specimen in the method of the present disclosure.Typically, " reference " can be obtained based on one or more normal specimens, particularly specimens that are not affected by cancer disease, obtained from a patient or one or more different individuals, for example, healthy individuals, particularly individuals of the same species." Reference " can be empirically determined by testing a sufficient number of normal specimens.

[0137] An "epitope" is a collective molecular feature, such as primary, secondary, and tertiary peptide structure, and charge, that together form a site recognized by, for example, an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor. Alternatively, an epitope can be defined as a set of amino acid residues involved in recognition by a particular immunoglobulin, or, from the perspective of T cells, as a set of residues 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" should be 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 a T cell, such as a T lymphocyte or a T helper cell. Epitopes can be prepared by isolation from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes may contain 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 may also be referred to in some cases as peptides or peptide epitopes. It should be understood that proteins or peptides comprising the epitopes or analogs described herein as well as additional amino acid(s) are still within the scope of this disclosure. In certain embodiments, peptides comprise fragments of antigens. In certain embodiments, there are limitations on the length of the peptides of the present disclosure. Length-restricted embodiments occur when a protein or peptide comprising an epitope described herein contains a region (i.e., a series of contiguous amino acid residues) that is 100% identical to a native sequence. For example, to avoid reading the definition of an epitope into terms of the entire native molecule, there is a limit on the length of any region that is 100% identical to 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 generally has 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 by a peptide having a region having fewer than 51 amino acid residues with 100% identity to the native peptide sequence in any increment down to 5 amino acid residues; for example, 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.

[0138] The nomenclature used to describe peptides or proteins follows conventional practice, with the amino group indicated at the left (amino- or N-terminus) of each amino acid residue and the carboxyl group at the right (carboxyl- or C-terminus). When amino acid residue positions are referred to in a peptide epitope, they are numbered from amino to carboxyl, with position 1 representing the residue located at the amino terminus of the epitope, or of the peptide or protein of which the epitope may be a part. In formulas illustrating selected specific embodiments of the present disclosure, the amino- and carboxyl-terminal groups are not specifically indicated, but are in the form they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally designated by a standard three-letter or single-letter designation. L-form amino acid residues are designated by a single capital letter or a three-letter designation with an initial capital letter, and the D-form for amino acid residues having the D-form is designated by a single lowercase letter or a three-letter designation with a lowercase letter. However, when three-letter designations or full names are used without capital letters, they may refer to L-amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or "G." The amino acid sequences of peptides presented herein are generally designated 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).

[0139] The term "mutation" refers to a change in a nucleic acid sequence or a difference in a nucleic acid sequence (nucleotide substitution, addition, or deletion) 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 children. 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. "Frameshift" occurs when a mutation disrupts the normal phase of the codon periodicity of a gene (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. Different mutations in a gene are possible to achieve the same altered reading frame.

[0140] " Conservative " amino acid substitution is the amino acid substitution in which one amino acid residue is replaced with another amino acid residue with similar side chain.The field has defined a family of amino acid residues with similar side chains, including 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 conservative substitutions of nucleotides and amino acids that do not eliminate peptide function are well known in the field.

[0141] As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, for example, between an HLA-binding peptide and 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 generic term to describe this chemical entity. It is a direct measure of the energy of binding. Affinity can be measured using, for example, commercially available Biacore Affinity can be determined experimentally by surface plasmon resonance (SPR) using an SPR unit. The affinity is determined by the inhibitory concentration 50 (IC), which is the concentration at which 50% of the peptide is displaced. 50 ) can also be expressed as ln(IC 50 ) is IC 50 It refers to the natural logarithm of K. off refers to the off-rate constant, for example, for the dissociation of an HLA-binding peptide and class I or II HLA. Throughout this disclosure, "binding data" results are referred to as "IC 50 " can be expressed in terms of IC 50 is the concentration of peptide tested 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., the limiting HLA protein and labeled reference peptide concentrations), these values ​​are D The binding activity of a peptide is approximately equal to the binding activity of the peptide. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publications WO94 / 20127 and WO94 / 03205, as well as other publications, such as 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 can be expressed relative to the binding of a reference standard peptide. For example, the IC of the reference standard peptide 50 Compared to the IC 50Binding can also be determined using other assay systems, including those using viable 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)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 21:2069 (1991)), and immunoassays using purified MHC (e.g., Hill et al., J. Immunol. 21:2069 (1991)). al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurements of class I MHC stabilization or assembly (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.

[0142] 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 synthesized according to standard protocols in the art. Synthetic epitopes can contain artificial amino acid residues, "amino acid mimetics," such as D-isomers of naturally occurring L-amino acid residues, or non-natural amino acid residues, such as cyclohexylalanine. Derived or prepared epitopes can be analogs of native epitopes.

[0143] "Native" or "wild-type" sequence refers to a sequence found in nature. Such a sequence may include a longer sequence found in nature.

[0144] "Receptor" should be understood to mean a biological molecule or group of molecules capable of binding to a ligand. Receptors can function to transmit information in cells, cell formations, or organisms. A receptor comprises at least one receptor unit, for example, where each receptor unit can be composed of a protein molecule. A receptor has a structure complementary to that of a ligand and can complex with the ligand as a binding partner. Information is transmitted, in particular, by a conformational change of the receptor after complexation of the ligand on the surface of a cell. In some embodiments, receptors should be understood to mean, in particular, MHC class I and II proteins capable of forming a receptor / ligand complex with a ligand, in particular, a peptide or peptide fragment of an appropriate length.

[0145] A "ligand" should be understood to mean a molecule that has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In some embodiments, a ligand should be understood to mean a peptide or peptide fragment of an appropriate length and containing an appropriate binding motif in its amino acid sequence, such that the peptide or peptide fragment is capable of forming a complex with an MHC class I or MHC class II protein.

[0146] In some embodiments, "receptor / ligand complex" should also be 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.

[0147] "Synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., man-made. Such peptides may be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion proteins."

[0148] The term "motif" refers to a pattern of residues in a peptide of a defined length, e.g., an amino acid sequence 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 a class I HLA motif, 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 a class II HLA motif, that is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in the pattern of primary and secondary anchor residues. In some embodiments, MHC class I motifs identify peptides that are 9, 10, or 11 amino acid residues in length.

[0149] The term "naturally occurring" and its grammatical equivalents, as used herein, refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in a laboratory, is naturally occurring.

[0150] 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 diseases. The term "individualized cancer vaccine" or "personalized cancer vaccine" refers to a specific cancer patient and means that the cancer vaccine is adapted to the needs or special circumstances of the individual cancer patient.

[0151] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially arrests disease symptoms, side effects, or progression. The immune response may also include an antibody response promoted by stimulation of helper T cells.

[0152] "Antigen processing" or "processing" and grammatical equivalents refer to the breakdown of a polypeptide or antigen into processing products that are fragments of that polypeptide or antigen (e.g., breakdown 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 by a cell, such as an antigen-presenting cell, to a specific T cell.

[0153] An "antigen-presenting cell" (APC) is a cell that presents peptide fragments of protein antigens in association with MHC molecules on its 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 displaying antigen fragments bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Additional costimulatory signals are then generated by the antigen-presenting cell, leading to T cell activation. The expression of costimulatory molecules is a defining feature of professional antigen-presenting cells. The major types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which have the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both MHC class II and class I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses, and activation of these cells is a critical step for the induction of antitumor immunity. Dendritic cells are conveniently categorized as "immature" and "mature" cells, which can be used as a simple method to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, 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 responsible for 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 4-1 BB).

[0154] The term "identical" and its grammatical equivalents, or "sequence identity," as used herein in terms of the amino acid sequences of two nucleic acid sequences or polypeptides, refers to residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window," as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200 contiguous positions, more usually about 100 to about 150 contiguous positions, within which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison is described in Smith et al. and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA, 85:2444 (1988); by computerized implementations of these algorithms, including, but not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA in the PC / Gene program by Intelligentics, Mountain View, Calif., and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is described in 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 visual 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 can also be described with reference to a starting nucleic acid, e.g., a nucleic acid can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% sequence identity to a reference nucleic acid or a fragment thereof, as measured, 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 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 find matching residues in the larger molecule according to the order in which the two molecules are optimally aligned.

[0155] The term "substantially identical" and its grammatical equivalents, when applied to a nucleic acid or amino acid sequence, means that the nucleic acid or amino acid sequence includes sequences having at least 90% or more, at least 95%, at least 98%, and at least 99% sequence identity compared to a reference sequence using standard parameters with a program 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)). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist 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 of sequence identity. In embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues long, over a region of at least about 100 residues, and in 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.

[0156] The term " vector " as used herein refers to the construct that can deliver one or more genes or sequences of interest and usually express them 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 associated with cationic condensing agent, and DNA or RNA expression vector encapsulated in liposome.

[0157] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include polypeptides, antibodies, polynucleotides, vectors, cells, or compositions that have been purified to a degree that they are no longer 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 that includes a polynucleotide amplified in the PCR or quantitative PCR reaction.

[0158] The terms "isolated," "biologically pure," or their grammatical equivalents refer to material that is substantially or essentially free from components that normally accompany that material when found in its native state. Thus, the isolated peptides described herein are free of some or all of the materials normally associated with the peptide in its in situ 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 does not have additional amino acid residues attached to it that result in a sequence with 100% identity over the entire length of the native sequence. The native sequence may be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term "isolated" means that the material has been removed from its original environment (e.g., the natural environment if it occurs in nature). An "isolated" nucleic acid is a nucleic acid that has been removed from its natural environment. For example, a naturally occurring polynucleotide or peptide present 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 can be part of a vector, and / or such polynucleotides or peptides can be part of a composition, and still be said to be "isolated" in that such vector or composition is not part of its 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.

[0159] The term "substantially purified" and its grammatical equivalents, as used herein, refers to a nucleic acid sequence, polypeptide, protein, or other compound that is substantially free, i.e., greater than about 50% free, greater than about 70% free, or greater than about 90% free, from polynucleotides, proteins, polypeptides, and other molecules with which it is naturally associated.

[0160] The term "substantially pure," as used herein, 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.

[0161] The terms "polynucleotide," "nucleotide," "nucleic acid," "polynucleic acid," or "oligonucleotide," and their grammatical equivalents, are used interchangeably herein to 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. This also includes modified forms of polynucleotides, e.g., by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also meant to encompass molecules containing non-naturally occurring or synthetic nucleotides and 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 can 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.

[0162] "Transfection," "transformation," or "transduction," as used herein, 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, including, for example, calcium phosphate DNA co-precipitation (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 co-precipitation (Brash et al., 2001). al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after growth of the infectious particles in suitable packaging cells, many of which are commercially available.

[0163] 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 may be called homologs. For example, any naturally occurring protein described herein can be modified by any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide 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 useful in establishing homology varies depending on the nucleic acid and protein in question, but as little as 25% sequence identity is routinely 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 sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are publicly available.

[0164] The term "subject" refers to any animal (e.g., mammal), including but not limited to, a human, a non-human primate, a dog, a cat, a rodent, etc., who will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0165] 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 occurrence of a disease or disorder; slow the development of a disease or disorder; slow the progression of a disease or disorder; alleviate to some extent one or more symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0166] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow, 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 development of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to having the disorder; and those in whom the disorder is to be prevented.

[0167] "Pharmaceutically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible.

[0168] A "pharmaceutical excipient" or "excipient" includes materials such as adjuvants, carriers, pH adjusting and buffering agents, osmolality adjusting agents, humectants, preservatives, etc. A "pharmaceutical excipient" is a pharmaceutically acceptable excipient. II. Neoantigens and Uses Thereof

[0169] One of the major obstacles to developing curative tumor-specific immunotherapy is the identification and selection of highly specific and restrictive 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 class of antigens.Due to the technical difficulties in identifying neoantigens, selecting optimized antigens, and producing neoantigens for use in vaccines or immunogenic compositions, neoantigens have rarely been used in cancer vaccines or immunogenic compositions. These problems may be addressed by: identifying mutations in neoplasms / tumors from a high percentage of subjects with cancer that are present at the DNA level in the tumor but not in matched germline samples; analyzing the identified mutations using one or more peptide-MHC binding prediction algorithms to generate multiple neo-antigenic T cell epitopes that are expressed within the neoplasm / tumor and bind to a high percentage of patient HLA alleles; and synthesizing multiple neo-antigenic peptides selected from the set of all neo-antigenic peptides and predicted binding peptides for use in cancer vaccines or immunogenic compositions suitable for treating a high percentage of subjects with cancer.

[0170] For example, converting peptide sequencing information into a therapeutic vaccine can involve predicting mutated peptides that can bind to HLA molecules in a high percentage of individuals. Effectively selecting specific mutations for use as immunogens requires the ability to predict which mutated peptides will efficiently bind to a high percentage of patients' HLA alleles. Recently, neural network-based learning approaches using validated binding and non-binding peptides have improved the accuracy of prediction algorithms for major HLA-A and HLA-B alleles. However, even with advanced neural network-based algorithms to encode HLA-peptide binding rules, several factors limit the ability to predict peptides presented on HLA alleles.

[0171] Another example of converting peptide sequencing information into a therapeutic vaccine could involve formulating a drug as a long peptide multi-epitope vaccine. Targeting as many mutated epitopes as practically possible harnesses the immune system's vast capabilities, prevents the chance of immunological escape due to down-modulation of immune-targeted gene products, and compensates for the known inaccuracy of epitope prediction approaches. Synthetic peptides provide a useful means for efficiently preparing multiple immunogens and rapidly converting the identification of mutated epitopes into effective vaccines. Peptides can be easily chemically synthesized and purified using reagents that are free of contaminating bacterial or animal substances. Their small size allows for unambiguous focus on the mutated region of a protein and also reduces irrelevant antigen competition from other components (non-mutated proteins or viral vector antigens).

[0172] Another example of converting peptide sequencing information into a therapeutic vaccine may involve combining it with a strong vaccine adjuvant. Effective vaccines may require a strong adjuvant to initiate an immune response. For example, poly-ICLC, an agonist of TLR3, and the RNA helicase domains of MDA5 and RIG3, exhibits 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 induces sustained CD4 activation in humans. + and CD8 + Importantly, striking similarities in the upregulation of transcriptional and signaling pathways were observed in subjects vaccinated with poly-ICLC and in volunteers who received a highly effective, replication-competent 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 +The peptide has been shown to induce T cell and antibody responses. At the same time, poly-ICLC has been extensively tested in more than 25 clinical trials to date and has demonstrated a relatively benign toxicity profile.

[0173] 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; a polynucleotide encoding the first peptide and the second peptide; one or more APCs comprising the first peptide and the second peptide; or a first T cell receptor (TCR) specific for the first neoepitope in a complex with an HLA protein and a second TCR specific for the 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.

[0174] 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; a polynucleotide encoding the first peptide and the second peptide; one or more APCs comprising the first peptide and the second peptide; or a first T cell receptor (TCR) specific for the first neoepitope in a complex with an HLA protein and a second TCR specific for the second neoepitope in a complex with an HLA protein; wherein the first peptide is different from the second peptide, and the first neoepitope comprises a first mutation and the second neoepitope comprises a second mutation.

[0175] 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 of a region of the same protein, and the second neoepitope comprises a second neoepitope of the same region of the same protein. In some embodiments, the first neoepitope and the second neoepitope comprise at least one amino acid of the same region. In some embodiments, this region of the 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 contiguous amino acids of the protein. In some embodiments, this 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 contiguous 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. 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 T cell's TCR is a class I receptor containing the first or second peptide. 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 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 sequences 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 column 2 of Table 1 or 2. In some embodiments, the mutation is a mutation in column 1 of Tables 3-14. In some embodiments, the protein is KRAS. 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 the sequence transcribed from the same transcription start site.In some embodiments, the first peptide is encoded by the sequence transcribed from the first transcription start site, and the second peptide is encoded by the sequence transcribed from the second transcription start site.In some embodiments, the 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. a first 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 a first sequence of 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. and a second sequence of at least 16 or 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 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 a sequence of at least 8 contiguous amino acids, wherein at least two of the 8 contiguous amino acids differ from 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 a sequence of at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from the corresponding positions in the wild-type sequence.

[0176] 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, and 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 higher 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 higher 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, the at least one anchor residue-adjacent region is upstream of the N-terminal anchor residue of the at least two anchor residues; downstream of the C-terminal anchor residue of the at least two anchor residues; or both (a) and (b).

[0177] 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 higher 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 50and binds to an HLA class II protein. In 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 50and binds to an HLA-peptide complex. 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 is a self-replicating 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-containing nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.

[0178] In some aspects, provided herein is a pharmaceutical composition comprising a composition described herein or a vector described herein; and a pharmaceutically acceptable excipient.

[0179] 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 Hiltonol.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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, the second peptide is administered to stimulate T cells, and the first peptide is administered after the second peptide to restimulate T cells. In some embodiments, the subject has cancer, the cancer being 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 treatment. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL that is resistant to ibrutinib treatment. 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, L792F, or C797S 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, the plurality of T cells comprising a first TCR and the 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 modality. In some embodiments, the at least one additional therapeutic agent or modality 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 administering the pharmaceutical composition described herein. III. Peptides

[0184] In embodiments, the present disclosure provides isolated peptides comprising the tumor-specific mutations of Tables 1-14. These peptides and polypeptides are referred to herein as "neo-antigenic peptides" or "neo-antigenic polypeptides." The polypeptides or peptides can be of various lengths, can be 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 polypeptides described herein. Table 1 [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 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 [Table 1-23] [Table 1-24] [Table 1-25] [Table 1-26] [Table 1-27] [Table 1-28] [Table 1-29] [Table 1-30] 1 The underlined AA indicates a non-native AA. 2 The bolded AA indicates the native AA of the amino acid sequence encoded by the second of the two fused genes. 3 The bold and underlined AA indicates a non-native AA in the amino acid sequence encoded by the second of the two fused genes due to a frameshift. Table 2 [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] 1 The underlined AA indicates a non-native AA. 2 The bolded AA indicates the native AA of the amino acid sequence encoded by the second of the two fused genes. 3 The bold and underlined AA indicates a non-native AA in the amino acid sequence encoded by the second of the two fused genes due to a frameshift.

[0185] In the above table, for one or more of the exemplary fusions, the sequence preceding the first ":" belongs to the exon sequence of the polypeptide encoded by the first gene, the sequence following the second ":" belongs to the exon sequence of the polypeptide encoded by the second gene, and the amino acids 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.

[0186] However, in some embodiments, e.g., NAB:STAT6, the NAB exon is joined to the 5'UTR of STAT6, and the first amino acid after the junction is the normal start codon for STAT6 (there is no frame at this site, as it is not normally translated).

[0187] In some embodiments, AR-V7 in the table above, a splice variant of the AR gene that encodes a protein lacking the ligand-binding domain found in the full-length AR, may also be considered.

[0188] In some embodiments, sequencing is used to identify tumor-specific mutations. Any suitable sequencing method, such as next-generation sequencing (NGS) technology, can be used in accordance with the present disclosure. Third-generation sequencing methods may replace NGS technology in the future to speed up the sequencing step of this 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 dividing the entire genome into small pieces, as opposed to the "traditional" sequencing methodology known as Sanger chemistry. Such NGS technology (also known as massively parallel sequencing technology) 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, and in principle may enable a single-cell sequencing approach. Several NGS platforms available commercially or mentioned in the literature, such as those described in detail in WO2012 / 159643, can be used in light of the present disclosure.

[0189] In certain embodiments, the peptides described herein have a nucleotide sequence similar to or similar 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 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 derivable range therein. In specific embodiments, the neo-antigenic peptide molecule is equal to or less than 100 amino acids.

[0190] In some embodiments, the peptide can be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. In some embodiments, the peptide can be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0191] In some embodiments, the 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.

[0192] In some embodiments, the peptides have a total 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.

[0193] In some embodiments, the peptides have a total 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.

[0194] Longer peptides can be designed in several ways. In some embodiments, when HLA-binding peptides are predicted or known, the longer peptides comprise (1) individual binding peptides with 2-5 amino acid extensions toward the N- and C-termini of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with their respective extended sequences. In other embodiments, if sequencing reveals long (>10 residues) neoepitope sequences present in the tumor (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), the longer peptides can consist of the entire novel tumor-specific stretch of amino acids, either as 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, resulting in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, where these peptides overlap and are spaced across the long neoantigenic peptide.

[0195] In some embodiments, the peptides 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 peptides may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the peptides may have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0196] In some embodiments, the peptides described herein may be in solution, lyophilized, or in crystalline form. In some embodiments, the peptides described herein may be synthetically prepared by recombinant DNA technology or chemical synthesis, or may be isolated from natural sources, such as native tumors or pathogenic organisms. Neoepitopes may be synthesized individually or may be directly or indirectly conjugated into the peptide. The peptides described herein may be substantially free of other naturally occurring host cell proteins and fragments thereof, although in some embodiments, the peptides may be synthetically conjugated to native fragments or particles.

[0197] In some embodiments, the peptides described herein can be prepared in a wide 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, individual peptides can be joined using chemical ligation to produce larger peptides that still fall within the scope of the present disclosure.

[0198] Alternatively, recombinant DNA technology can be used in which a nucleotide sequence encoding the peptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultivated under appropriate conditions 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 containing one or more neo-antigenic peptides described herein can be used to present appropriate T cell epitopes.

[0199] 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 are possible to achieve 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 with 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, and the peptide comprises an amino acid sequence encoded by an out-of-frame sequence resulting from the fusion.

[0200] In some aspects, the present disclosure provides compositions comprising at least two or more peptides. In some embodiments, the compositions described herein comprise at least two distinct peptides. In some embodiments, the compositions described herein comprise 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 vary in length, amino acid sequence, or both. The peptides may be derived from any protein known to contain or found to contain a tumor-specific mutation. 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 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.

[0201] In some embodiments, peptides can be derived from proteins with substitution mutations, such as KRAS G12C, G12D, G12V, Q61H, or Q61L mutations, or NRAS Q61K or Q61R 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 are located 2-5 residues from the N-terminus or 2-5 residues from the C-terminus. Peptides can also be derived from tumor-specific insertion mutations, where the peptide includes one or more, or all, of the inserted residues.

[0202] 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.

[0203] 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. a first 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 a first sequence of 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. 1 sequence; and a second sequence of at least 16 or 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.

[0204] 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.

[0205] In some embodiments, the first peptide, the second peptide, or both, comprise at least one flanking sequence, and this 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, hi some embodiments, at least one flanking residue comprises a mutation.

[0206] 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 minimum 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 minimum 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; 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.

[0207] In some embodiments, the peptide comprises a neo-antigenic peptide sequence shown in Tables 1-14. In some embodiments, the peptide comprises a neo-epitope sequence shown in Tables 1-14. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least one mutant amino acid (underlined amino acid) shown in Tables 1-14. 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) shown in Tables 1-14. In some embodiments, the peptide comprises a neo-epitope sequence comprising at least one mutant amino acid (underlined amino acid) and at least one bolded amino acid shown in Tables 1-14. In some embodiments, the peptide comprises a neoepitope sequence derived from a protein that includes at least one mutant amino acid (underlined amino acid) shown in Tables 1-14 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 (underlined amino acid) shown in Tables 1-14 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 minimum 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) shown in Tables 1-14 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 minimum 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) shown in Tables 1-14, 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 minimum 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 minimum one mutant amino acid.

[0208] 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 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%, 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 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%, 99% or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the 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 the corresponding wild-type sequence upstream of at least one mutant amino acid, at least one mutant amino acid. and a neoepitope sequence derived from a protein comprising a 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%, 99% or 100% sequence identity to the corresponding wild-type sequence downstream of at least one mutant amino acid.

[0209] In some embodiments, the peptide has a sequence similar to that of at least one mutant amino acid and 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%. and a neoepitope sequence derived from a protein comprising a sequence containing 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 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the peptide has a sequence similar to that of at least one mutant amino acid and 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%. and a neoepitope sequence derived from a protein comprising a sequence containing 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 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.In some embodiments, the 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%, 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%, 166%, 167%, Contains 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 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity sequence, and 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%, 166%, 167%, 168 and a neoepitope sequence derived from a protein comprising a sequence containing 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 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0210] In some embodiments, the peptide comprises a neoepitope sequence derived from a protein comprising at least one mutant amino acid (an underlined amino acid) shown in Tables 1-14 and a sequence upstream of the at least one mutant amino acid 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%, 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) shown in Tables 1-14 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) shown in Tables 1-14, 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 sequence upstream of the one mutant amino acid. % sequence identity to the corresponding wild-type sequence downstream of at least one mutant amino acid, and a neoepitope sequence derived from a protein comprising a 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%, 99% or 100% sequence identity to the corresponding wild-type sequence downstream of at least one mutant amino acid.

[0211] In some embodiments, the peptide has at least one mutant amino acid (underlined amino acid) shown in Tables 1-14 and 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% of the corresponding wild-type sequence upstream of the mutant amino acid. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a protein comprising a sequence containing 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. In some embodiments, the 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%, 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%, 166%, 167%, , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a protein comprising a sequence containing 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.In some embodiments, the peptide comprises at least one mutant amino acid (underlined amino acid) shown in Tables 1-14, 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%, 1 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 with 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity a sequence containing consecutive amino acids and 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 downstream of at least one mutant amino acid; , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a protein comprising a sequence containing 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.

[0212] In some embodiments, the peptide comprising a KRAS G12C mutation comprises the sequence MTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In some embodiments, the peptide comprising a KRAS G12C mutation comprises the neoepitope sequence of KLVVVGACGV. In some embodiments, the peptide comprising a KRAS G12C mutation comprises the neoepitope sequence of LVVVGACGV. In some embodiments, the peptide comprising a KRAS G12C mutation comprises the neoepitope sequence of VVGACGVGK. In some embodiments, the peptide comprising a KRAS G12C mutation comprises the neoepitope sequence of VVVGACGVGK.

[0213] In some embodiments, the peptide comprising a KRAS G12D mutation comprises the sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In some embodiments, the peptide comprising a KRAS G12D mutation comprises the neoepitope sequence of VVGADGVGK. In some embodiments, the peptide comprising a KRAS G12D mutation comprises the neoepitope sequence of VVVGADGVGK. In some embodiments, the peptide comprising a KRAS G12D mutation comprises the neoepitope sequence of KLVVVGADGV. In some embodiments, the peptide comprising a KRAS G12D mutation comprises the neoepitope sequence of LVVVGADGV.

[0214] In some embodiments, the peptide comprising a KRAS G12V mutation comprises the sequence MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. In some embodiments, the peptide comprising a KRAS G12V mutation comprises a neoepitope sequence of KLVVVGAVGV. In some embodiments, the peptide comprising a KRAS G12V mutation comprises a neoepitope sequence of LVVVGAVGV. In some embodiments, the peptide comprising a KRAS G12V mutation comprises a neoepitope sequence of VVGAVGVGK. In some embodiments, the peptide comprising a KRAS G12V mutation comprises a neoepitope sequence of VVVGAVGVGK.

[0215] In some embodiments, the peptide comprising the KRAS Q61H mutation comprises the sequence: AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. In some embodiments, the peptide comprising the KRAS Q61H mutation comprises the neoepitope sequence of ILDTAGHEEY.

[0216] In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the neoepitope sequence of ILDTAGLEEY. In some embodiments, a peptide comprising a KRAS Q61L mutation comprises the neoepitope sequence of LLDILDTAGL.

[0217] In some embodiments, the peptide comprising the NRAS Q61K mutation comprises the sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. In some embodiments, the peptide comprising the NRAS Q61K mutation comprises the neoepitope sequence of ILDTAGKEEY.

[0218] In some embodiments, the peptide comprising the NRAS Q61R mutation comprises the sequence: AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. In some embodiments, the peptide comprising the NRAS Q61R mutation comprises the neoepitope sequence of ILDTAGREEY.

[0219] In some embodiments, a peptide comprising a mutation in the TMPRSS2:ERG fusion comprises the neoepitope sequence of MALNS::EALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDWALNSEALSV. In some embodiments, a peptide comprising a mutation in the TMPRSS2:ERG fusion comprises the neoepitope sequence of ALNSEALSVV. In some embodiments, a peptide comprising a mutation in the TMPRSS2:ERG fusion comprises the neoepitope sequence of MALNSEALSV. In some embodiments, a peptide comprising a mutation in the RAS Q61H mutation comprises the sequence of TCLLDILDTAGHEEYSAMRDQYM.

[0220] In some embodiments, the peptide comprising the RAS Q61H mutation comprises a sequence provided in Table 3. In some embodiments, the peptide sequence provided in Table 3 binds or is predicted to bind to a protein encoded by an HLA allele, the allele being provided in the corresponding column in Table 3 next to the peptide sequence. Table 3. Peptide sequences containing the RAS Q61H mutation, corresponding HLA alleles, and ranking of binding potential. [Table 3-1] [Table 3-2]

[0221] In some embodiments, the peptide comprising the RAS Q61R mutation comprises the sequence TCLLDILDTAGREEYSAMRDQYM. In some embodiments, the peptide comprising the RAS Q61R mutation comprises a sequence provided in Table 4. In some embodiments, the peptide sequences provided in Table 4 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 4 next to the peptide sequence. Table 4. Peptide sequences containing the RAS Q61R mutation, corresponding HLA alleles, and ranking of binding potential. [Table 4-1] [Table 4-2]

[0222] In some embodiments, the peptide comprising the RAS Q61K mutation comprises the sequence TCLLDILDTAGKEEYSAMRDQYM. In some embodiments, the peptide comprising the RAS Q61K mutation comprises a sequence provided in Table 5. In some embodiments, the peptide sequences provided in Table 5 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 5 next to the peptide sequence. Table 5. Peptide sequences containing the RAS Q61K mutation, corresponding HLA alleles, and ranking of binding potential. [Table 5-1] [Table 5-2]

[0223] In some embodiments, the peptide comprising the RAS Q61L mutation comprises the sequence TCLLDILDTAGLEEYSAMRDQYM. In some embodiments, the peptide comprising the RAS Q61L mutation comprises a sequence provided in Table 6. In some embodiments, the peptide sequences provided in Table 6 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 6 next to the peptide sequence. Table 6. Peptide sequences containing the RAS Q61L mutation, corresponding HLA alleles, and ranking of binding potential. [Table 6-1] [Table 6-2]

[0224] In some embodiments, the peptide comprising the RAS G12A mutation comprises the sequence MTEYKLVVVGAAGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12A mutation comprises a sequence provided in Table 7. In some embodiments, the peptide sequences provided in Table 7 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 7 next to the peptide sequence. Table 7. Peptide sequences containing the RAS G12A mutation, corresponding HLA alleles, and ranking of binding potential. [Table 7-1] [Table 7-2]

[0225] In some embodiments, the peptide comprising the RAS G12C mutation comprises the sequence MTEYKLVVVGACGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12C mutation comprises a sequence provided in Table 8. In some embodiments, the peptide sequences provided in Table 8 bind or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 8 adjacent to the peptide sequence. Table 8. Peptide sequences containing the RAS G12C mutation, corresponding HLA alleles, and ranking of binding potential. [Table 8-1] [Table 8-2]

[0226] In some embodiments, the peptide comprising the RAS G12D mutation comprises the sequence MTEYKLVVVGADGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12D mutation comprises a sequence provided in Table 9. In some embodiments, the peptide sequences provided in Table 9 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 9 next to the peptide sequence. Table 9. Peptide sequences containing the RAS G12D mutation, corresponding HLA alleles, and ranking of binding potential. [Table 9-1] [Table 9-2]

[0227] In some embodiments, the peptide comprising the RAS G12R mutation comprises the sequence MTEYKLVVVGARGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12R mutation comprises a sequence provided in Table 10. In some embodiments, the peptide sequences provided in Table 10 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 10 adjacent to the peptide sequence. Table 10. Peptide sequences containing the RAS G12R mutation, corresponding HLA alleles, and ranking of binding potential [Table 10-1] [Table 10-2]

[0228] In some embodiments, the peptide comprising the RAS G12S mutation comprises the sequence MTEYKLVVVGASGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12S mutation comprises a sequence provided in Table 11. In some embodiments, the peptide sequences provided in Table 11 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 11 adjacent to the peptide sequence. Table 11. Peptide sequences containing the RAS G12S mutation, corresponding HLA alleles, and ranking of binding potential [Table 11-1] [Table 11-2]

[0229] In some embodiments, the peptide comprising the RAS G12V mutation comprises the sequence MTEYKLVVVGAVGVGKSALTIQL. In some embodiments, the peptide comprising the RAS G12V mutation comprises a sequence provided in Table 12. In some embodiments, the peptide sequences provided in Table 12 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 12 adjacent to the peptide sequence. Table 12. Peptide sequences containing the RAS G12V mutation, corresponding HLA alleles, and ranking of binding potential [Table 12-1] [Table 12-2]

[0230] In some embodiments, the peptide comprising the RAS G13C mutation comprises the sequence MTEYKLVVVGAGCVGKSALTIQL. In some embodiments, the peptide comprising the RAS G13C mutation comprises a sequence provided in Table 13. In some embodiments, the peptide sequences provided in Table 13 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 13 adjacent to the peptide sequence. Table 13. Peptide sequences containing the RAS G13C mutation, corresponding HLA alleles, and ranking of binding potential [Table 13-1] [Table 13-2]

[0231] In some embodiments, the peptide comprising the RAS G13D mutation comprises the sequence MTEYKLVVVGAGDVGKSALTIQL. In some embodiments, the peptide comprising the RAS G13D mutation comprises a sequence provided in Table 14. In some embodiments, the peptide sequences provided in Table 14 bind to or are predicted to bind to proteins encoded by HLA alleles, the alleles being provided in the corresponding column in Table 14 adjacent to the peptide sequence. Table 14. Peptide sequences containing the RAS G13D mutation, corresponding HLA alleles, and ranking of binding potential [Table 14-1] [Table 14-2] A. Peptide Modification

[0232] In some embodiments, the present disclosure includes modified peptides. Modifications may include covalent chemical modifications that do not alter the primary amino acid sequence of the antigenic peptide itself. Modifications may produce peptides with desirable properties, such as extending in vivo half-life, increasing stability, reducing clearance, altering immunogenicity or allergenicity, enabling the elicitation of specific antibodies, cellular targeting, antigen uptake, antigen processing, HLA affinity, HLA stability, or antigen presentation. In some embodiments, the peptide may include one or more sequences that enhance epitope processing and presentation by APCs, for example, for generating an immune response.

[0233] In some embodiments, peptides can be modified to provide 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 T helper cell response. In some embodiments, immunogenic peptide / T helper conjugates are linked by a spacer molecule. In some embodiments, the spacer comprises a relatively small, neutral molecule, such as an amino acid or amino acid mimetic, that is substantially uncharged under physiological conditions. The spacer can be selected, for example, from Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It is 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 T helper peptide directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the neo-antigenic peptide or the T helper peptide can be acylated. Examples of T helper peptides include tetanus toxoid residues 830-843, influenza residues 307-319, and malaria circumsporozoite residues 382-398 and residues 378-389.

[0234] The peptide sequences of the present disclosure can be altered, if desired, through changes at the DNA level, particularly by mutating the DNA encoding the peptide at preselected bases to generate codons that translate into the desired amino acids.

[0235] In some embodiments, the peptides described herein may contain substitutions that alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, peptides may be modified by substitution of cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​has a propensity to form disulfide bridges, structurally altering peptides sufficiently to reduce binding capacity. Substituting C with α-aminobutyric acid not only alleviates this problem, but may actually improve binding and cross-linking capacity in certain cases. Substitution of cysteine ​​with α-aminobutyric acid may occur at any residue of 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.

[0236] Peptides can also be modified by, for example, adding or deleting amino acids to extend or reduce the amino acid sequence of the compound.Peptides or analogs can also be modified by changing the order or composition of certain residues.It is understood by those skilled in the art that certain amino acid residues important for biological activity, such as amino acid residues at important contact sites or conserved residues, generally may not be altered without adverse effects on biological activity.Non-essential amino acids need not be limited to those naturally occurring in proteins, such as L-α-amino acids or their D-isomers, but can also include unnatural amino acids, such as β-γ-δ-amino acids, as well as many derivatives of L-α-amino acids.

[0237] In some embodiments, peptides can be modified using a series of peptides with single amino acid substitutions to determine the effect of electrostatic 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 exhibit different patterns of sensitivity to various HLA molecules and T cell receptors. Additionally, multiple substitutions using small, relatively neutral moieties, such as Ala, Gly, Pro, or similar residues, can be used. Substitutions can be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the required spacing between essential contact points and a particular functional property desired (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for HLA molecules or T cell receptors compared to the affinity of the parent peptide can also be achieved by such substitutions. In either event, such substitutions should use amino acid residues or other molecular fragments selected to avoid, for example, steric and charge interferences that could disrupt binding. Amino acid substitutions are typically single-residue substitutions. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide.

[0238] In some embodiments, the peptides described herein contain amino acid mimetics or unnatural amino acid residues, for example, D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; 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 D-(aryl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine, whose alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, or isopentyl, or non-acidic amino acid residues. Aromatic rings of unnatural amino acids 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.

[0239] In some embodiments, the peptides described herein are acylated by terminal -NH2 acylation, e.g., alkanoyl (C1-C 20) or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia, methylamine, etc. In some embodiments, these modifications may provide sites for linkage to a support or other molecule. In some embodiments, the peptides described herein may include modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surface-active materials, e.g., lipids, or may be chemically modified, e.g., acetylation. Furthermore, bonds in peptides may be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0240] In some embodiments, the peptides described herein may include a carrier, such as those well known in the art, for example, thyroglobulin, albumin, such as human serum albumin, tetanus toxoid, polyamino acid residues, such as poly-L-lysine and poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.

[0241] 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 peptides and the like. A review 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 provide certain desired properties, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide, which binds to the desired HLA molecule and activates appropriate T cells. For example, peptides can be subject to various changes, e.g., either conservative or non-conservative substitutions, which can provide certain advantages in their use, e.g., improved HLA binding. Such conservative substitutions can involve replacing an amino acid residue with another that is biologically and / or chemically similar, for example, replacing one hydrophobic residue with another, or one polar residue with another. The effect of single amino acid substitutions can also be explored using D-amino acids. Such modifications can be made, for example, as described by 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 This can be done using well-known peptide synthesis procedures such as those described in Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[0242] In some embodiments, the peptides described herein may 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., toxoids from diphtheria, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells.

[0243] Modifications to the peptide can include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle conjugation, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant material, addition of an amino acid mimetic, or addition of a non-natural amino acid.

[0244] Glycosylation can affect the physical properties of proteins and may also be important for protein stability, secretion, and subcellular localization. Proper glycosylation can be important for biological activity. Indeed, when some genes from eukaryotes are expressed in bacteria (e.g., E. coli) that lack the cellular processes for glycosylation of proteins, the lack of glycosylation results in recovered proteins with little or no activity. 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 commonly found on both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides, and 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 achieved chemically or enzymatically, or by substituting the codon encoding the amino acid residue to be glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endoglycosidases and exoglycosidases.

[0245] Further 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 above.

[0246] Another type of modification is the conjugation (e.g., linking) of one or more additional components or molecules, such as another protein (e.g., a protein having an amino acid sequence heterologous to 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 other components or molecules. In some embodiments, fusion of albumin to a peptide or protein of the present disclosure can be achieved by genetic engineering, such as by joining a DNA encoding HSA or a fragment thereof to a DNA encoding one or more polypeptide sequences. A suitable host can then be transformed or transfected with the fused nucleotide sequence, for example, in the form of a suitable plasmid, to express the fusion polypeptide. Expression can be achieved in vitro, for example, from prokaryotic or eukaryotic cells, or in vivo, for example, from transgenic organisms. In some embodiments of the present disclosure, expression of the fusion protein is carried out in a mammalian cell system, for example, 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; 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 protein therapeutic drugs.

[0247] Further candidate components and molecules for conjugation include components and molecules 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.Purification methods, such as cation exchange chromatography, can be used to separate conjugates by charge differences, which effectively separate 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.

[0248] 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 allowing the biopharmaceutical product to require less frequent administration. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, protecting the Fc fusion molecule from degradation and allowing it to be re-released into the circulation, maintaining the molecule in circulation for longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to traditional Fc fusion conjugates.

[0249] 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 involves modifying the peptide sequence by hydroxyethyl starch modification, which utilizes hydroxyethyl starch derivatives linked to other molecules to modify the characteristics of the molecule.Various embodiments of hydroxyethyl starch modification are described, for example, in US Patent Application Publication No. 2007 / 0134197 and US Patent Application Publication No. 2006 / 0258607.

[0250] Peptide stability can be assayed in several 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 appropriate 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.

[0251] Problems associated with short plasma half-lives or susceptibility 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 polyoxyalkylenes (e.g., typically via linking moieties covalently attached to both the protein and the nonproteinaceous polymer, e.g., PEG; see, for example). Such PEG-conjugated biomolecules have been shown to have clinically useful properties, including better physical and thermal stability, protection from susceptibility to enzymatic degradation, increased solubility, longer in vivo circulatory half-lives and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

[0252] PEGs suitable for conjugation to polypeptide or protein sequences are generally soluble in water at room temperature and have the general formula R—(O—CH—CH) nThe PEG has the formula -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, R generally has 1 to 8 carbons. The PEG conjugated to the 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, where the PEGs have different n values, and thus various different PEGs are present in specific ratios. For example, some compositions include mixtures of conjugates, where n=1, 2, 3, and 4. In some compositions, the percentage of conjugates where n=1 is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is at most 5%. 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 attached PEGs can be identified and purified to be free of the unmodified protein sequence and conjugates with other numbers of attached PEGs.

[0253] 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 bond between PEG and a free amino or carboxyl group of one or more polypeptide sequences. PEGs having a spacer that can be attached to a free amino group 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 a free amino group 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.

[0254] Conjugation of one or more peptide or protein sequences of the present disclosure to a spacer-bearing PEG can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out using a 4:1 to 30:1 molar ratio of reagents to peptide / protein in a solution 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 to predominantly 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, while high temperatures, neutral to high pH (e.g., pH > 7), and longer reaction times tend to increase the number of PEGs attached. Various means known in the art can be used to terminate the reaction. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing, for example, at -20°C.

[0255] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the properties of PEG (e.g., enhanced serum half-life) while imparting several additional advantageous properties. For example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser, and Thr) capable of forming an extended conformation similar to PEG can be recombinantly produced already fused to the peptide or protein drug of interest (e.g., Amunix XTEN Technology; Mountain View, CA). This eliminates the need for an additional conjugation step during the manufacturing process. Furthermore, established molecular biology techniques allow for control of the side chain composition of the polypeptide chain, enabling optimization of immunogenicity and manufacturing properties. B. Neoepitope

[0256] Neoepitopes include neoantigenic peptides or neoantigenic polypeptides recognized by the immune system. Neoepitopes refer to epitopes that are absent in 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 due to one or more mutations in diseased cells, e.g., cancer cells, the sequence of the epitope is altered to create a neoepitope. The term "neoepitope" is used interchangeably herein with "tumor-specific neoepitope" to designate a series of residues, typically L-amino acids, connected to each other by peptide bonds, typically between the α-amino and carboxyl groups of adjacent amino acids. Neoepitopes can be of various lengths, can be in either their neutral (uncharged) or salt form, and can contain or be free of modifications, such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptides described herein. The present disclosure provides isolated neoepitopes comprising the tumor-specific mutations of Tables 1-14.

[0257] 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.

[0258] 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 read-through mutation, a gene fusion mutation, and any combination thereof.

[0259] 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 the class I HLA-peptide complex.

[0260] 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 8-12 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 16-25 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 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 the corresponding positions in the wild-type sequence.

[0261] 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.

[0262] 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.

[0263] In some embodiments, the second neoepitope or both comprise 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).

[0264] 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 with higher affinity than the corresponding wild-type peptide. In some embodiments, the neoepitope has an IC of less than 5,000 nM, less than 1,000 nM, less than 500 nM, less than 100 nM, less than 50 nM, or even less. 50 It has.

[0265] In some embodiments, the neoepitope may have an HLA binding affinity of between about 1 pM and about 1 mM, between about 100 pM and about 500 μM, between about 500 pM and about 10 μM, between about 1 nM and about 1 μM, or between about 10 nM and about 1 μM. In some embodiments, the neoepitope may have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, or 1,000 nM, or greater. In some embodiments, the neoepitope may have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, or 1,000 nM.

[0266] In some embodiments, the first and / or second neoepitope binds to an HLA protein with higher affinity than the corresponding wild-type neoepitope, hi some embodiments, the first and / or second neoepitope has a K of less than 1,000 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 1,000 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 an HLA class I protein with a K of less than 2,000 nM, 1,500 nM, 1,000 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 and binds to HLA class II proteins.

[0267] In one embodiment, the first and / or second neoepitope binds to a protein encoded by an HLA allele expressed by the subject. In another embodiment, the mutation is not present in the subject's non-cancer cells. In yet another embodiment, the first and / or second neoepitope is encoded by a gene or expressed gene in the subject's cancer cells.

[0268] In some embodiments, the first neoepitope comprises a mutation shown in column 2 of Table 1 or 2 or column 1 of Tables 3-14. In some embodiments, the second neoepitope comprises a mutation shown in column 2 of Table 1 or 2 or column 1 of Tables 3-14. In some embodiments, the first neoepitope and the second neoepitope are derived from a TMPRSS2:ERG fusion protein. In some embodiments, the first neoepitope and the second neoepitope are derived from a TMPRSS2:ERG fusion protein comprising the sequence S::E from the sequence MALNS::EALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDWALNSEALSV. For example, the first neoepitope and the second neoepitope may comprise the sequence ALNSEALSVV. For example, the first neoepitope and the second neoepitope can comprise the sequence MALNSEALSV.

[0269] In some embodiments, the first neoepitope and the second neoepitope are derived from a KRAS protein. In some embodiments, the first neoepitope and the second neoepitope are derived from an NRAS protein. In some embodiments, the first neoepitope and the second neoepitope are derived from a KRAS protein comprising a G12C, G12D, G12V, Q61H, or Q61L substitution mutation. In some embodiments, the first neoepitope and the second neoepitope are derived from an NRAS protein comprising a Q61K or Q61R substitution mutation. In some embodiments, the neoepitope comprises a substitution mutation, for example, a KRAS G12C, G12D, G12V, Q61H, or Q61L mutation, or an NRAS Q61K or Q61R mutation. In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence MTEYKLVVVGACGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. For example, the first and second neoepitopes may comprise the sequence KLVVVGACGV. For example, the first and second neoepitopes may comprise the sequence LVVVGACGV. For example, the first and second neoepitopes may comprise the sequence VVGACGVGK. For example, the first and second neoepitopes may comprise the sequence VVVGACGVGK. In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEVVGADGVGK. For example, the first and second neoepitopes may comprise the sequence VVVGADGVGK. For example, the first and second neoepitopes may comprise the sequence KLVVVGADGV. For example, the first and second neoepitopes may comprise the sequence LVVVGADGV.

[0270] In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQE. For example, the first and second neoepitopes may comprise the sequence KLVVVGAVGV. For example, the first and second neoepitopes may comprise the sequence LVVVGAVGV. For example, the first and second neoepitopes may comprise the sequence VVGAVGVGK. For example, the first and second neoepitopes may comprise the sequence VVVGAVGVGK.

[0271] In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. For example, the first and second neoepitopes may comprise the sequence ILDTAGHEEY.

[0272] In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGLEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM. For example, the first and second neoepitopes may include the sequence ILDTAGLEEY. For example, the first and second neoepitopes may include the sequence LLDILDTAGL.

[0273] In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGKEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. For example, the first and second neoepitopes may comprise the sequence ILDTAGKEEY.

[0274] In some embodiments, the first and second neoepitopes are derived from the KRAS or NRAS protein sequence AGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGREEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPM. For example, the first and second neoepitopes may comprise the sequence ILDTAGREEY.

[0275] In some embodiments, the neoepitope is selected from the group consisting of DTAGHEEY, TAGHEEYSAM, DILDTAGHE, DILDTAGH, ILDTAGHEE, ILDTAGHE, DILDTAGHEEY, DTAGHEEYS, LLDILDTAGH, DILDTAGRE, DILDTAGR, ILDTAGREE, ILDTAGRE, CLLDILDTAGR, TAGREEYSAM, REEYSAMRD, DTAGKEEYSAM, CLLDILDTAGK, DTAGKEEY, LLDILDTAGK, ILDTAGKE, ILDTAGKEE, DTAG LEEY, ILDTAGLE, DILDTAGL, ILDTAGLEE, GLEEYSAMRDQY, LLDILDTAGLE, LDILDTAGL, DILDTAGLE, DILDTAGLEEY, AGVGKSAL, GAAGVGKSAL, AAGVGKSAL, CGVG KSAL, ACGVGKSAL, DGVGKSAL, ADGVGKSAL, DGVGKSALTI, GARGVGKSA, KLVVVGARGV, VVVGARGV, SGVGKSAL, VVVGASGVGK, GASGVGKSAL, VGVGKSAL, VVVGAGCVGK , KLVVVGAGC, GDVGKSAL, DVGKSALTI, VVVGAGDVGK, TAGKEEYSAM, DTAGHEEYSAM, TAGHEEYSA, DTAGREEYSAM, TAGKEEYSA, AAGVGKSA, AGCVGKSAL, AGDVGKSAL ,AGKEEYSAMR,AGVGKSALTI,ARGVGKSAL,ASGVGKSA,ASGVGKSAL,AVGVGKSA,CVGKSALTI,DILDTAGK,DILDTAGREEY,DTAGHEEYSAMR,DTAGKEEYS,DTAGKEEYS AMR, DTAGLEEYS, DTAGLEEYSA, DTAGLEEYSAMR, DTAGREEYS, DTAGREEYSAMR, GAAGVGKSA, GACGVGKSA, GACGVGKSAL, GADGVGKS, GAGDVGKSA, GAGDVGKSAL, GA SGVGKSA, GCVGKSAL, GCVGKSALTI, GHEEYSAM, GKEEYSAM, GLEEYSAMR, GREEYSAM, GREEYSAMR, HEEYSAMRD, KEEYSAMRD, KLVVVGASG, LDILDTAGR, LEEYSAMRD,LVVVGARGV, LVVVGASGV, REEYSAMRDQY, RGVGKSAL, TAGLEEYSA, TEYKLVVVGAA, VGAAGVGKSA, VGADGVGK, VGASGVGKSA, VGVGKSALTI, VVVGAAGV, VVVGAVGV, YKLVVVGAC, YKLVVVGAD, YKLVVVGAR, and DILDTAGKE.

[0276] The substitutions can be located anywhere along the length of the neoepitope. 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 another embodiment, the substituted residues are located 2-5 residues from the N-terminus or 2-5 residues from the C-terminus. Peptides can also be derived from tumor-specific insertion mutations, where the peptide contains one or more, or all, of the inserted residues.

[0277] In some embodiments, the peptides described herein can be readily chemically synthesized using reagents free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). In some embodiments, peptides are prepared by (1) parallel solid-phase synthesis on a multichannel instrument using homogeneous synthesis and cleavage conditions; (2) purification on an RP-HPLC column with column stripping and re-cleaning, but without substitution between peptides; followed by (3) analysis using a limited set of the most informative assays. Good manufacturing practice (GMP) footprints can be defined around the set of peptides for individual patients, thus requiring only a single set of conversion procedures between the synthesis of peptides for different patients. C. Polynucleotides

[0278] Alternatively, nucleic acids (e.g., polynucleotides) encoding the peptides of the present disclosure can be used to produce neo-antigenic peptides in vitro. The polynucleotides can be, for example, DNA, cDNA, PNA, CNA, RNA, either single-stranded and / or double-stranded, or native or stabilized forms of polynucleotides, such as polynucleotides with phosphorothioate backbones, or combinations thereof, and may or may not contain introns, so long as they encode the peptide. In some embodiments, in vitro translation is used to produce the peptide.

[0279] Neoantigenic polynucleotides encoding each of the neoantigenic peptides described in this disclosure are provided herein. The terms "polynucleotide," "nucleotide," or "nucleic acid" are used interchangeably in this disclosure with "mutant polynucleotide," "mutant nucleotide," "mutant nucleic acid," "neoantigenic polynucleotide," "neoantigenic nucleotide," or "neoantigenic mutant nucleic acid." Various nucleic acid sequences can encode the same peptide due to redundancy in the genetic code. Each of these nucleic acids is within the scope of this disclosure. The nucleic acid encoding the peptide can be DNA or RNA, e.g., mRNA, or a combination of DNA and RNA. In some embodiments, the nucleic acid encoding the peptide is a self-replicating mRNA (Brito et al., Adv. Genet. 2015; 89:179-233). Any suitable polynucleotide encoding the peptides described herein is within the scope of this disclosure.

[0280] The term "RNA" includes "mRNA" and, in some embodiments, relates to "mRNA." The term "mRNA" means "messenger-RNA" and refers to a "transcript" encoding a peptide or polypeptide, produced using a DNA template. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In some embodiments, mRNA is a self-replicating mRNA. In view of the present disclosure, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0281] The stability and translation efficiency of RNA can be modified as needed. For example, RNA can be stabilized and its translation can be increased by one or more modifications that have a stabilizing effect and / or increase the translation efficiency of RNA. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of the RNA used in accordance with the present disclosure, it can be modified in the coding region, i.e., the sequence encoding the peptide or protein to be expressed, without changing the sequence of the peptide or protein to be expressed, to increase mRNA stability and to perform codon optimization, thereby increasing GC content to enhance translation in cells.

[0282] The term "modified," in terms of RNA as used in this disclosure, includes any modification of RNA that does not naturally occur in that RNA. In some embodiments, the RNA does not have an uncapped 5'-triphosphate. Removal of such an uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase. In other embodiments, the RNA can have modified ribonucleotides to increase its stability and / or reduce cytotoxicity. In some embodiments, 5-methylcytidine can, for example, partially or completely replace cytidine in the RNA. Alternatively, pseudouridine, for example, partially or completely replaces uridine.

[0283] In some embodiments, the term "modification" refers to providing an RNA with a 5'-cap or a 5'-cap analog. The term "5'-cap" refers to the cap structure found on the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via an unusual 5'-5' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7 position. The term "conventional 5'-cap" refers to the naturally occurring RNA 5'-cap, the 7-methylguanosine cap (mG). In the context of the present disclosure, the term "5'-cap" includes 5'-cap analogs that have been modified to resemble the RNA cap structure and have the ability to stabilize RNA and / or enhance translation of RNA when bound thereto in vivo and / or in cells.

[0284] In certain embodiments, mRNA encoding the neo-antigenic peptide of the present disclosure is administered to a subject in need thereof. In some embodiments, the present disclosure provides RNA, oligoribonucleotide and polyribonucleotide molecules comprising modified nucleosides, gene therapy vectors comprising the same, gene therapy methods and gene transcription silencing methods comprising the same. In some embodiments, the administered mRNA comprises at least one modified nucleoside.

[0285] Polynucleotides encoding the peptides described herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides that comprise or consist of analogs can be simply made by substituting the appropriate desired nucleobase(s) for the nucleobase(s) encoding the native epitope.

[0286] The polynucleotides described herein may contain one or more synthetic or naturally occurring introns in the transcribed region.The inclusion of mRNA stabilizing sequences and sequences for replication in mammalian cells may also be considered to increase polynucleotide expression.In addition, the polynucleotides described herein may contain immunostimulatory sequences (ISS or CpG).These sequences may be included in vectors outside the polynucleotide coding sequence to enhance immunogenicity.

[0287] In some embodiments, a polynucleotide may include a coding sequence for a peptide or protein fused in the same reading frame to a polynucleotide that aids in the expression and / or secretion of the peptide or protein from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide from the cell). A polypeptide with a leader sequence may be a preprotein, having a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[0288] In some embodiments, the polynucleotide may contain a peptide or protein coding sequence fused in the same reading frame to a marker sequence that allows for purification of the encoded peptide, which can then be incorporated into, for example, a personalized disease vaccine or immunogenic composition. For example, in the case of a bacterial host, the marker sequence may be a hexahistidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker, or, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, Xpress tag, Isopep tag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose-binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, etc.

[0289] In some embodiments, a polynucleotide may contain coding sequences for one or more of the peptides or proteins described herein fused in the same reading frame to create a single concatemerized neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0290] In some embodiments, DNA sequences are constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. If necessary, the sequence can be mutagenized by site-directed mutagenesis to provide a functional analog thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585. In another embodiment, a DNA sequence encoding a peptide or protein of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired peptide and by selecting codons preferred in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0291] Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the particular isolated polypeptide of interest is inserted into an expression vector and, if necessary, operably linked to appropriate expression control sequences for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene can be operably linked to transcriptional and translational expression control sequences functional in the selected expression host.

[0292] Accordingly, the present disclosure also relates to vectors and expression vectors useful for the production and administration of the neo-antigenic peptides and neoepitopes described herein, as well as host cells containing such vectors. IV. Vector

[0293] In some embodiments, an expression vector capable of expressing the peptides or proteins described herein can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into the host bacterium for cloning using standard techniques (e.g., Sambrook (See, e.g., J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0294] Numerous vectors and host systems suitable for producing and administering the neo-antigenic peptides described herein are known to those of skill in the art and are commercially available. The following vectors are provided as examples: Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pCR (Invitrogen). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.

[0295] For expression of the neo-antigenic peptides described herein, the coding sequence is provided with operably linked start and stop codons, a promoter and terminator region, and, in some embodiments, a replication system to provide an expression vector for expression in a desired cellular host. For example, a promoter sequence compatible with a bacterial host is provided in a plasmid containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vector is transformed into a suitable bacterial host.

[0296] Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. Such promoters can also be derived from viral sources, such as human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type 1 (HSV TK promoter). Nucleic acid sequences derived from SV40 splice and polyadenylation sites can be used to provide the required nontranscribed genetic elements.

[0297] Recombinant expression vectors can be used to amplify and express DNA encoding the peptides or proteins described herein. A recombinant expression vector is a replicable DNA construct containing a synthetic or cDNA-derived DNA fragment encoding a peptide or biologically equivalent analog operably linked to appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally contains an assembly of (1) genetic elements that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences, as described in detail herein. Such regulatory elements may include an operator sequence to control transcription. An origin of replication and the ability to replicate in a host, usually conferred by a selection gene to facilitate recognition of transformants, may also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue which, if desired, can be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0298] Generally, recombinant expression vectors contain an origin of replication and a selectable marker, such as the ampicillin resistance gene in E. coli and the TRP1 gene in S. cerevisiae, that allows transformation of host cells, as well as a promoter derived from a highly expressed gene that directs transcription of downstream structural sequences. Such promoters can be derived from operons encoding glycolytic enzymes, such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences and, in some embodiments, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein containing an N-terminal identification peptide that confers desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product.

[0299] Polynucleotides encoding the neo-antigenic peptides described herein may also include a ubiquitination signal sequence and / or a targeting sequence, for example, an endoplasmic reticulum (ER) signal sequence, which facilitates translocation of the resulting peptide into the endoplasmic reticulum.

[0300] In some embodiments, the neo-antigenic peptides described herein can also be administered and / or expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences encoding the neo-antigenic peptides described herein. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is Bacillus Calmette-Guerin (BCG). BCG vectors are described by Stover et al., Nature 351:456-460 (1991).

[0301] A wide variety of other vectors useful for therapeutic administration or immunization of the neoantigenic polypeptides described herein will be apparent to those skilled in the art from the disclosure herein, including adeno- and adeno-associated viral vectors, retroviral vectors, Salmonella Typhimurium vectors, detoxified anthrax toxin vectors, Sendai viral vectors, pox viral vectors, canarypox vectors, and fowlpox vectors. In some embodiments, the vector is Modified Vaccinia Ankara (VA) (e.g., Bavarian Noridic (MVA-BN)).

[0302] Vectors that can be used in the practice of the present disclosure allow integration into the host genome of cells via retroviral gene transfer, often resulting in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Retroviruses can also be engineered to enable conditional expression of the inserted transgene, so that lentiviruses infect only certain cell types. Cell-type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore, both lentiviral and retroviral vectors can be used in the practice of the present disclosure). Furthermore, lentiviral vectors can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with the capacity to package up to 6-10 kb of foreign sequence. A minimal set of cis-acting long terminal repeats is sufficient for replication and packaging of the vector, which is then used to integrate the desired nucleic acid into target cells to provide persistent expression.Widely used retroviral vectors that can be used in the practice of the present disclosure include retroviral vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfelt et al., (1990) Virol. 176:58-59; Wilson et al., (1998) J. Virol. 63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224; PCT / US94 / 05700).

[0303] Minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV), are also useful in practicing the present disclosure. These vectors may have a cytomegalovirus (CMV) promoter driving the expression of target genes. Thus, the present disclosure contemplates viral vectors, including retroviral and lentiviral vectors, among other vectors useful in practicing the present disclosure.

[0304] Adenovirus vectors are also useful in implementing the present disclosure.One advantage is the ability of recombinant adenovirus to efficiently transfer and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of transferred nucleic acid.In addition, the ability to productively infect quiescent cells expands the usefulness of recombinant adenovirus vectors.Furthermore, high expression level ensures that the nucleic acid product is expressed at a level sufficient to generate an immune response (see, for example, U.S. Patent No. 7,029,848, which is hereby incorporated by reference).

[0305] For adenovirus vectors useful in the practice of the present disclosure, reference is made to U.S. Patent No. 6,955,808. The adenovirus vector used can be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome has been published (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents of which are hereby incorporated by reference). Ad35 vectors are described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors are described in U.S. Patent No. 6,913,922. C6 adenovirus vectors are described in U.S. Patent Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are described in U.S. Patent No. 6,277,558. E1-deleted or deleted, E3-deleted or deleted, and / or E4-deleted or deleted adenovirus vectors can also be used. E1-deleted adenovirus mutants are replication-deficient in non-permissive cells, or at least highly attenuated, so certain adenoviruses with mutations in the E1 region have an improved safety margin. Adenoviruses with mutations in the E3 region may have enhanced immunogenicity by disrupting the mechanism by which adenovirus downregulates MHC class I molecules.Adenoviruses with E4 mutations may have reduced immunogenicity of adenoviral vectors due to the suppression of late gene expression.Such vectors may be particularly useful when repeated revaccination using the same vector is desired.Adenoviral vectors having deletions or mutations in E1, E3, E4; E1 and E3; and E1 and E4 can be used in accordance with the present disclosure.

[0306] Furthermore, "gutless" adenoviral vectors, in which all viral genes have been deleted, can also be used in accordance with the present disclosure. Such vectors require a helper virus for replication and require a special human 293 cell line that expresses both E1a and Cre, a state not present in the natural environment. Such "gutless" vectors are non-immunogenic, and therefore, the vectors can be administered multiple times for revaccination. "Gutless" adenoviral vectors can be used for the insertion of heterologous inserts / genes, such as the transgenes of the present disclosure, and can even be used for the co-delivery of multiple heterologous inserts / genes.

[0307] In some embodiments, delivery is via adenovirus, which may be a single booster dose. In some embodiments, adenovirus is delivered via multiple doses. For in vivo delivery, AAV is advantageous over other viral vectors due to its low toxicity and low likelihood of causing insertional mutagenesis due to its lack of integration into the host genome. AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb result in significantly reduced virus production. There are many promoters that can be used to drive nucleic acid molecule expression. AAV ITRs can function as promoters, which is advantageous because they eliminate the need for additional promoter elements.

[0308] For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For expression in the brain, the following promoters can be used: Synapsin I for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include Pol III promoters, such as U6 or H1. The use of Pol II promoters and intron cassettes can be used to express guide RNAs (gRNAs). For the AAV vectors useful in the implementation of the present disclosure, reference is made to U.S. Patent Nos. 5,658,785, 7,115,391, 7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540, 6,475,769 and 6,258,595 and the documents cited therein.For AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof.AAV can be selected according to the cell to be targeted; for example, for targeting brain or neuronal cells, AAV serotype 1, 2, 5, or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected; for targeting cardiac tissue, AAV4 can be selected.AAV8 is useful for delivery to the liver. In some embodiments, delivery is via AAV. Dosage can be adjusted to balance the therapeutic benefit against any side effects.

[0309] In some embodiments, poxviruses are used in the compositions described herein. These include orthopoxvirus, avian pox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, and the like (see, e.g., Verardi et al., Hum. Vaccin. Immunother. 2012 Jul;8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development and research in many fields. Advantages of these vectors include simple construction, the ability to accommodate large amounts of foreign DNA, and high expression levels. Information regarding poxviruses that can be used in the practice of the present disclosure, such as, inter alia, poxviruses of the subfamily Chordopoxvirinae (vertebrate poxviruses), e.g., orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth strain, WR strain (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 strain, ALVAC), fowlpox virus (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, their synthetic or non-naturally occurring recombinant forms, their uses, and methods for making and using such recombinants, can be found in the scientific and patent literature.

[0310] In some embodiments, vaccinia virus is used in disease vaccines or immunogenic compositions to express antigens (Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr. Opin. Immunol. 9:517-524, (1997). Recombinant vaccinia viruses can replicate within the cytoplasm of infected host cells, thus allowing the polypeptide of interest to induce an immune response. Furthermore, poxviruses are widely used as vaccine or immunogenic composition vectors due to their ability to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, particularly antigen-presenting cells, but also due to their ability to self-adjuvant.

[0311] In some embodiments, ALVAC is used as a vector in disease vaccines or immunogenic compositions. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol. Immunother. 2000;49:504-14; von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin. Cancer. Res. 2000; 6:2219-28;Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals. J. Immunol. 2003;171:1094-101; Paoletti E. Applications of pox virus vectors to vaccination: an update. Proc. Natl. Acad. Sci. USA 1996;93:11349-53; U.S. Patent No. 7,255,862). In a phase I clinical trial, ALVAC virus expressing the tumor antigen CEA showed a favorable safety profile and generated increased CEA-specific T cell responses in selected patients; however, no objective clinical responses were observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J. Clin. Oncol. 1999;17:332-7).

[0312] In some embodiments, modified vaccinia Ankara (MVA) virus can be used as a viral vector for antigen vaccines or immunogenic compositions. MVA is a member of the Orthopoxvirus family and was generated by serial passage of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts approximately 570 times (see, for example, Mayr, A., et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus contains 31 kilobases less genomic information than CVA and is highly host cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, i.e., reduced toxicity or infectivity, yet retains excellent immunogenicity. When tested in various animal models, MVA has proven to be non-toxic, even in immunosuppressed individuals. Furthermore, MVA-BN®-HER2 is a candidate immunotherapy designed for the treatment of HER-2-positive breast cancer and is currently undergoing clinical trials (Mandl et al. al., Cancer Immunol. Immunother. Jan 2012; 61(1): 19-29). Methods for making and using recombinant MVA have been described (see, e.g., U.S. Patent Nos. 8,309,098 and 5,185,146, which are hereby incorporated in their entireties).

[0313] Suitable host cells for expressing polypeptides include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0314] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell systems include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5'- or 3'-flanking non-transcribed sequences, and 5'- or 3'-untranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0315] Host cells are genetically engineered (transduced or transformed or transfected) with a vector, which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a viral particle, a phage, etc. The engineered host cells may be cultured in conventional nutrient media modified as necessary to activate promoters, select transformants, or amplify the polynucleotide. Culture conditions, e.g., temperature, pH, etc., will be those previously used in the host cell chosen for expression and will be apparent to those of skill in the art.

[0316] Representative examples of suitable hosts include bacterial cells, such as E. coli, Bacillus subtilis, Salmonella typhimurium, and various species of the genera Pseudomonas, Streptomyces, and Staphylococcus; fungal cells, such as yeast; insect cells, such as Drosophila and Sf9; animal cells, such as monkey kidney fibroblasts of the COS-7 system described by Gluzman, Cell 23:175 (1981), as well as other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines, or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the skill of one skilled in the art from the teachings herein.

[0317] Yeast, insect, or mammalian cell hosts can also be used using appropriate vectors and control sequences. Examples of mammalian expression systems include the COS-7 monkey kidney fibroblast line described by Gluzman, Cell 23:175 (1981), as well as other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines.

[0318] The polynucleotides described herein can be administered and expressed in human cells (e.g., immune cells, including dendritic cells). A human codon usage table can be used to guide codon selection for each amino acid. Such polynucleotides can include spacer amino acid residues between epitopes and / or analogs, such as those described above, or can include epitopes and / or analogs (and / or CTLs (e.g., CD8 + ), Th (e.g., CD4 + ) and B-cell epitopes).

[0319] Standard regulatory sequences well known to those skilled in the art can be included in the vector to ensure expression in human target cells. Several vector elements are desirable: a promoter with a downstream cloning site for inserting a polynucleotide, such as a minigene; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Many promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. For other suitable promoter sequences, see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466. In some embodiments, the promoter is a CMV-IE promoter.

[0320] Useful expression vectors for eukaryotic hosts, particularly mammalian or human, include, for example, vectors containing expression control sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, e.g., plasmids derived from Escherichia coli, including pCR1, pBR322, pMB9, and their derivatives, and broader host range plasmids, e.g., M13 and filamentous single-stranded DNA phages.

[0321] Vectors can be introduced into animal tissues by several different methods. The two most common approaches are injection of DNA in saline using a standard hypodermic needle, and gene gun delivery. A general overview of the construction of DNA vaccine plasmids and their subsequent delivery into the host by these two methods is provided in Scientific American (Weiner et al., (1999) Scientific American 281 (1): 34-41). Injection in saline solution is usually performed intramuscularly (IM) in skeletal muscle or intradermally (ID), and DNA is delivered to the extracellular space. This can be assisted by electroporation, by temporarily damaging muscle fibers with myotoxins, such as bupivacaine; or by using a hypertonic solution of saline or sucrose (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). The immune response to this method of delivery can be affected by many factors, including the needle type, needle alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the animal being injected (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410).

[0322] Another commonly used method of delivery, gene gun delivery, uses compressed helium as an accelerator to ballistically accelerate plasmid DNA (pDNA) adsorbed onto gold or tungsten microparticles into target cells (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0323] Alternative delivery methods may include aerosol instillation of naked DNA onto mucosal surfaces, such as the nasal and pulmonary mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the ocular and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88). Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA can also be delivered to cells after gentle mechanical disruption of the cell membrane, which transiently permeabilizes the cells. Such gentle mechanical disruption of the membrane can be achieved by gently passing the cells through a small opening (Sharei et al., Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Systems, vol. 1, no. 1). Cells, PLOS ONE (2015)).

[0324] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. "Liposome" is a generic term encompassing a variety of mono- and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess amount of aqueous solution. The lipid components undergo self-reorganization before forming closed structures, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions having structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0325] The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with a lipid, combined with a lipid, contained in a suspension in a lipid, contained within or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in plasma, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0326] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar. Lipids, Inc. (Birmingham, Ala.) Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about −20° C. Chloroform is used as the only solvent because it evaporates more readily than methanol.

[0327] In some embodiments, the vector comprises a polynucleotide encoding 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 vary in length, amino acid sequence, or both. The peptides are derived from any protein known to contain or found to contain a tumor-specific mutation. In some embodiments, the vector comprises 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 vector comprises 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 read-through mutation, a gene fusion mutation, and any combination thereof.

[0328] In some embodiments, the vector comprises a polynucleotide operably linked to a promoter. In some embodiments, the vector is a self-replicating RNA replicon, a plasmid, a phage, a transposon, a cosmid, a virus, or a virion. 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-containing nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere. VT cell receptor

[0329] In one aspect, the present disclosure provides cells that express neo-antigen-recognizing receptors (e.g., T cell receptors (TCRs) or chimeric antigen receptors (CARs)) that activate immunoresponsive cells, and methods of using such cells for the treatment of diseases requiring an enhanced immune response. Such cells include genetically engineered immunoresponsive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs (e.g., CD8 + )) cells, helper T lymphocytes (Th (e.g., CD4 + )) cells) and methods of their use for the treatment of neoplasms and other pathologies in which an increase in antigen-specific immune response is desired. T cell activation is mediated by TCRs or CARs targeted to antigens.

[0330] The present disclosure provides cells expressing a combination of antigen-recognizing receptors (e.g., TCRs, CARs) and chimeric costimulatory receptors (CCRs) that activate immunoresponsive cells, and methods of using such cells for the treatment of diseases requiring an enhanced immune response. In some embodiments, tumor antigen-specific T cells, NK cells, CTL cells, or other immunoresponsive cells are used as shuttles for the selective enrichment of one or more costimulatory ligands for the treatment or prevention of neoplasms. Such cells are administered to a human subject in need thereof for the treatment or prevention of certain cancers.

[0331] In some embodiments, tumor antigen-specific human lymphocytes that may be used in the methods of the present disclosure include, without limitation, peripheral donor lymphocytes genetically modified to express chimeric antigen receptors (CARs) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45), peripheral donor lymphocytes genetically modified to express the full-length tumor antigen-recognizing T cell receptor complex, including the a and p heterodimers (Morgan, RA, et al. 2006 Science 314:126-129), lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies (Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 J Immunol 164:4382-4392), and in vivo immunophenotyping using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells. These include antigen-specific peripheral blood leukocytes selectively expanded in vitro (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood 102:2498-2505). T cells can be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

[0332] In some embodiments, the immunotherapeutic agent is an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a B cell receptor (BCR), adoptive T cell therapy (ACT), or a derivative thereof. In other aspects, the engineered receptor is a chimeric antigen receptor (CAR). In some aspects, the CAR is a first-generation CAR. In other aspects, the CAR is a second-generation CAR. In still other aspects, the CAR is a third-generation CAR. In some aspects, the CAR comprises an extracellular portion, a transmembrane portion, and an intracellular portion. In some aspects, the intracellular portion comprises at least one T cell costimulatory domain. In some embodiments, the T cell costimulatory domain is selected from the group consisting of CD27, CD28, TNFRS9(4-1BB), TNFRSF4(OX40), TNFRSF8(CD30), CD40LG(CD40L), ICOS, ITGB2(LFA-1), CD2, CD7, KLRC2(NKG2C), TNFRS18(GITR), TNFRSF14(HVEM), or any combination thereof.

[0333] In some embodiments, the engineered receptor binds to a target, hi some embodiments, the binding is specific to a peptide specific for one or more subjects suffering from a disease or condition.

[0334] In some aspects, the immunotherapeutic agent is a cell as described in detail herein. In some aspects, the immunotherapeutic agent is a cell comprising a receptor that specifically binds to a peptide or neoepitope described herein. In some aspects, the immunotherapeutic agent is a cell used in combination with the peptide / nucleic acid of the present disclosure. In some embodiments, the cell is a patient cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte.

[0335] In some aspects, a subject with a condition or disease is treated based on the subject's T cell receptor repertoire. In some embodiments, a peptide or neoepitope is selected based on the subject's T cell receptor repertoire. In some embodiments, the subject is treated with T cells expressing a TCR specific for a peptide or neoepitope described herein. In some embodiments, the subject is treated with a peptide or neoepitope specific for a TCR, e.g., a subject-specific TCR. In some embodiments, the subject is treated with a peptide or neoepitope specific for a TCR, e.g., a T cell expressing a subject-specific TCR. In some embodiments, the subject is treated with a peptide or neoepitope specific for a subject-specific TCR.

[0336] In some embodiments, the compositions described herein are selected based on TCRs identified in one or more subjects. In some embodiments, identifying the T cell repertoire and testing in a functional assay is used to determine the composition to administer to one or more subjects with a condition or disease. In some embodiments, the composition is an antigen vaccine comprising one or more peptides or proteins described herein. In some embodiments, the vaccine comprises a subject-specific neoantigenic peptide. In some embodiments, the peptides included in the vaccine are selected based on quantification of subject-specific TCRs that bind to the neoepitope. In some embodiments, the peptides are selected based on the binding affinity of the peptide to the TCR. In some embodiments, the selection is based on a combination of both quantity and binding affinity. For example, because T cells expressing the TCR are advantageously amplified, a TCR that strongly binds to the neoepitope in a functional assay but is not highly represented in the TCR repertoire may be a good candidate for an antigen vaccine.

[0337] In some embodiments, peptides or proteins are selected for administration to one or more subjects based on binding to a TCR. In some embodiments, T cells, e.g., T cells from a subject with a disease or condition, can be expanded. Expanded T cells expressing a TCR specific for the neoantigenic peptide or neoepitope can be administered back to the subject. In some embodiments, appropriate cells, e.g., PBMCs, are transduced or transfected with a polynucleotide for expression of a TCR specific for the neoantigenic peptide or neoepitope and administered to the subject. T cells expressing a TCR specific for the neoantigenic peptide or neoepitope can be expanded and administered back to the subject. In some embodiments, T cells expressing a TCR specific for the neoantigenic peptide or neoepitope that produce cytolytic activity when incubated with autologous diseased tissue can be expanded and administered to the subject. In some embodiments, T cells used in functional assays that produce binding to the neoantigenic peptide or neoepitope can be expanded and administered to the subject. In some embodiments, a TCR determined to bind to a subject-specific neoantigenic peptide or neoepitope can be expressed in a T cell and administered to a subject.

[0338] In certain embodiments, the present disclosure provides a composition comprising a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope, 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 provided herein comprise a first T cell comprising a first T cell receptor (TCR) specific for the first neoepitope and a second T cell comprising a second TCR specific for the second neoepitope. In some embodiments, the first and second peptides are derived from the same protein.

[0339] In another embodiment, the present disclosure provides a composition comprising 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 compositions provided herein comprise a first T cell comprising a first T cell receptor (TCR) specific for the first neoepitope and a second T cell comprising a second TCR specific for the second neoepitope. In some embodiments, the first mutation and the second mutation are the same.

[0340] 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 first 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 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 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 the class I HLA-peptide complex.

[0341] In some embodiments, the first TCR is a first chimeric antigen receptor specific for a first neoepitope, and the second TCR is a second chimeric antigen receptor specific for a second neoepitope. In some embodiments, the first T cell is a cytotoxic T cell. In some embodiments, the first T cell is a gamma delta T cell. In some embodiments, the second T cell is a helper T cell. In some embodiments, the first and / or second TCR has a K of less than 1,000 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 TCR binds to an HLA-peptide complex with a K of less than 1,000 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 TCR binds to an HLA class I-peptide complex with a K of less than 2,000, 1,500, 1,000 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 and binds to HLA class II-peptide complexes. VI. Antigen presenting cells

[0342] The neo-antigenic peptides or proteins may be provided as antigen-presenting cells (e.g., dendritic cells) comprising the peptides, proteins, or polynucleotides described herein. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. Accordingly, one embodiment of the present disclosure is a composition comprising at least one antigen-presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neo-antigenic peptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with the neo-antigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient. In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with the neo-antigenic peptides or nucleic acids. The neo-antigenic peptides can be any suitable peptide that generates an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides derived from tumor-associated antigens is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278. In some embodiments, the T cells are CTLs (e.g., CD8 + In some embodiments, the T cells are helper T lymphocytes (Th (e.g., CD4 + )).

[0343] In some embodiments, the present disclosure provides compositions comprising cell-based immunogenic pharmaceutical compositions that can also be administered to a subject. For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any of the well-known techniques, carriers, and excipients that are suitable and understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, the APC-based immunogenic pharmaceutical composition can be a dendritic cell-based immunogenic pharmaceutical composition.

[0344] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared via ex vivo or in vivo methods. Ex vivo methods can include the use of autologous DCs pulsed ex vivo with the polypeptides described herein to activate or load DCs before administration to patients. In vivo methods can include targeting specific DC receptors using antibodies coupled with the polypeptides described herein. DC-based immunogenic pharmaceutical compositions can further include DC activators, such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions can further include an adjuvant and a pharmaceutically acceptable carrier.

[0345] Antigen-presenting cells (APCs) can be prepared from various sources, including humans and non-human primates, other mammals, and vertebrates. In certain embodiments, APCs can be prepared from the blood of humans or non-human vertebrates. APCs can also be isolated from enriched populations of leukocytes. The leukocyte population can be prepared by methods known to those skilled in the art. Such methods typically include heparinized blood collection, apheresis or leukopheresis, buffy coat preparation, rosette formation, centrifugation, density gradient centrifugation (e.g., using Ficoll, colloidal silica particles, and sucrose), differential lysis of non-leukocyte cells, and filtration. The leukocyte population can also be prepared by collecting blood from a subject, defibrinating it to remove platelets, and lysing red blood cells. The leukocyte population can optionally be enriched for monocytic dendritic cell precursors.

[0346] Blood cell population can be obtained from various subjects according to the desired use of enriched leukocyte population.The subject can be a healthy subject.Alternatively, blood cells can be obtained from subjects that require immune stimulation, such as cancer patients or other patients that benefit from immune stimulation.Similarly, blood cells can be obtained from subjects that require immunosuppression, such as patients with autoimmune disorders (e.g., rheumatoid arthritis, diabetes, lupus, multiple sclerosis, etc.).Leukocyte population can also be obtained from HLA-matched healthy individuals.

[0347] When blood is used as a source of APCs, blood leukocytes can be obtained using conventional methods that maintain their viability. According to one embodiment of the present disclosure, blood can be diluted in culture medium, which may or may not contain heparin or other suitable anticoagulants. The blood to culture medium volume ratio can be approximately 1:1. Cells can be concentrated by centrifugation of the blood in culture medium at approximately 1,000 rpm (150 g) at 4°C. Platelets and red blood cells can be depleted by resuspending the cells in any number of solutions known in the art to lyse red blood cells, such as ammonium chloride. For example, the mixture can be a 1:1 volume ratio of culture medium and ammonium chloride. The cells can be concentrated by centrifugation and washed in the desired solution until a population of white blood cells substantially free of platelets and red blood cells is obtained. Any isotonic solution commonly used in tissue culture can be used as a medium for separating blood leukocytes from platelets and red blood cells. Examples of such isotonic solutions include phosphate-buffered saline, Hank's balanced salt solution, and complete growth medium. APCs and / or APC precursor cells can also be purified by elutriation.

[0348] In one embodiment, APCs can be non-nominal APCs under inflammatory or other activated conditions. For example, non-nominal APCs can include epithelial cells stimulated with interferon-gamma, T cells, B cells, and / or monocytes activated by factors or conditions that induce APC activity. Such non-nominal APCs can be prepared according to methods known in the art.

[0349] APCs can be cultured, expanded, differentiated, and / or matured as desired, depending on the type of APC. APCs can be cultured in any suitable culture vessel, such as, for example, a culture plate, a flask, a culture bag, or a bioreactor.

[0350] In certain embodiments, APCs can be cultured in an appropriate culture or growth medium to maintain and / or expand the number of APCs in the preparation. The culture medium can be selected according to the type of APC to be isolated. For example, mature APCs, such as mature dendritic cells, can be cultured in an appropriate growth medium for their maintenance and expansion. The culture medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. Additionally, cytokines, growth factors, and / or hormones can be included in the growth medium. For example, cytokines such as granulocyte / macrophage colony-stimulating factor (GM-CSF), FMS-like tyrosine kinase 3 ligand (FLT-3L), and / or interleukin 4 (IL-4) can be added for the maintenance and / or expansion of mature dendritic cells. In other embodiments, immature APCs can be cultured and / or expanded. Immature dendritic cells can retain the ability to take up target mRNA and process new antigens. In some embodiments, immature dendritic cells can be cultured in an appropriate medium for their maintenance and culture. The culture medium may be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. Additionally, cytokines, growth factors and / or hormones may be included in the growth medium.

[0351] Other immature APCs can be similarly cultured or expanded. Preparations of immature APCs can be matured to form mature APCs. APC maturation can occur during or after exposure to neoantigenic peptides. In certain embodiments, preparations of immature dendritic cells can be matured. Suitable maturation factors include, for example, the cytokine TNF-α, bacterial products (e.g., BCG), etc. In another aspect, isolated APC precursors can be used to prepare preparations of immature APCs. The APC precursors can be cultured, differentiated, and / or matured. In certain embodiments, monocytic dendritic cell precursors can be cultured in the presence of an appropriate culture medium supplemented with amino acids, vitamins, cytokines, and / or divalent cations to promote the differentiation of the monocytic dendritic cell precursors into immature dendritic cells. In some embodiments, the APC precursors are isolated from PBMCs. PBMCs can be obtained from a donor, e.g., a human donor, and can be used fresh or frozen for future use. In some embodiments, the APC is prepared from one or more APC preparations. In some embodiments, the APC comprises APCs loaded with a first and a second neo-antigenic peptide comprising a first and a second neo-epitope or a polynucleotide encoding a first and a second neo-antigenic peptide comprising a first and a second neo-epitope. In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC.

[0352] In certain embodiments, the present disclosure provides a composition comprising an APC comprising a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope, 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 first and second peptides are derived from the same protein. In another embodiment, the present disclosure provides a composition comprising an APC comprising 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. VII. Adjuvants

[0353] Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in patients receiving the compositions provided herein. Sometimes, adjuvants can elicit a Th1-type response. Sometimes, adjuvants can elicit a Th2-type response. A Th1-type response can be characterized by the production of cytokines such as IFN-γ, in contrast to a Th2-type response, which can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0354] In some embodiments, lipid-based adjuvants, such as MPLA and MDP, can be used in the immunogenic pharmaceutical compositions disclosed herein.For example, monophosphoryl lipid A (MPLA) is an adjuvant that causes increased presentation of liposomal antigens to specific T lymphocytes.In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in conjunction with the immunogenic pharmaceutical preparations described herein.

[0355] Suitable adjuvants are known in the art (see WO2015 / 095811), and include poly(I:C), poly-ICLC, Hiltonol, STING agonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, FLT-3L, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide Adjuvants include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® Vector System, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants also include Freund's incomplete or GM-CSF. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparations have been previously described (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev. Biol. Stand. 1998; 92:3-11) (Mosca et al. Frontiers in Bioscience, 2007; 12:4050-4060) (Gamvrellis et al. Immunol & Cell Biol. 2004; 82: 506-516). Cytokines can also be used. Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, FLT-3L, PGE1, PGE2, IL-1, IL-1b, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, the entire contents of which are incorporated herein by reference), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J. Immunother. Emphasis Tumor Immunol. 1996 (6):414-418).

[0356] Adjuvants can also include stimulatory molecules, such as cytokines. Non-limiting examples of cytokines include: CCL20, alpha-interferon (IFN-a), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ (lymphotoxin alpha (LTα)), GM-CSF, FLT-3L, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant form of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor. Condition, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK 2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0357] Additional adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor , fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR 5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and their functional fragments.

[0358] In some embodiments, the adjuvant may be a toll-like receptor modulator. Examples of toll-like receptor modulators include TLR-9 agonists, but are not limited to small molecule modulators of toll-like receptors, such as imiquimod. Other examples of adjuvants used in combination with the immunogenic pharmaceutical compositions described herein may include, but are not limited to, saponins, CpG ODNs, and the like. Sometimes, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may include at least one oil and at least one surfactant, and the oil(s) and surfactant(s) are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can be less than 5 μm in diameter, or even submicron in diameter; these small sizes are achieved using a microfluidizer to provide a stable emulsion. Droplets with a size less than 220 nm can be subjected to sterilization by filtration. VIII. METHODS OF TREATMENT AND PHARMACEUTICAL COMPOSITIONS

[0359] The neo-antigen therapeutics described herein (e.g., peptides, polynucleotides, TCRs, CARs, cells comprising a TCR or CAR, APCs or dendritic cells comprising a polypeptide, dendritic cells comprising a polynucleotide, antibodies, etc.) are useful in a variety of applications, including, but not limited to, therapeutic treatment methods, e.g., cancer treatment. In some embodiments, therapeutic treatment methods include immunotherapy. In certain embodiments, the neo-antigenic peptides are useful for activating, promoting, increasing, and / or enhancing an immune response, redirecting an existing immune response against a new target, increasing the immunogenicity of a tumor, inhibiting tumor growth, reducing tumor volume, increasing tumor cell apoptosis, and / or reducing the tumorigenicity of a tumor. Methods of use can be in vitro, ex vivo, or in vivo methods.

[0360] In some aspects, the present disclosure provides methods for activating an immune response in a subject using the neo-antigenic peptides or proteins described herein. In some embodiments, the present disclosure provides methods for promoting an immune response in a subject using the neo-antigenic peptides described herein. In some embodiments, the present disclosure provides methods for increasing an immune response in a subject using the neo-antigenic peptides described herein. In some embodiments, the present disclosure provides methods for enhancing an immune response using the neo-antigenic peptides. In some embodiments, the activation, promotion, increase, and / or enhancement of the immune response comprises increasing cell-mediated immunity. In some embodiments, the activation, promotion, increase, and / or enhancement of the immune response comprises increasing T cell activity or humoral immunity. In some embodiments, the activation, promotion, increase, and / or enhancement of the immune response comprises increasing CTL or Th activity. In some embodiments, the activation, promotion, increase, and / or enhancement of the immune response comprises increasing NK cell activity. In some embodiments, the activation, promotion, increase, and / or enhancement of the immune response comprises increasing T cell activity and increasing NK cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises increasing CTL activity and increasing NK cell activity. In some embodiments, activating, promoting, increasing, and / or enhancing the immune response comprises inhibiting or decreasing the suppressive activity of T regulatory (Treg) cells. In some embodiments, the immune response is the result of antigenic stimulation. In some embodiments, the antigenic stimulation is tumor cells. In some embodiments, the antigenic stimulation is cancer.

[0361] In some embodiments, the present disclosure provides methods of a...

Claims

[Claim 1] The invention described in the specification.

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