COMPOSITIONS AND METHODS FOR PREPARING T CELL COMPOSITIONS AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 15, 2020, is named 50401-744(Generic)_SL.txt and is 313,317 bytes in size.

BACKGROUND

Adoptive immunotherapy or adoptive cellular therapy with lymphocytes (ACT) is the transfer of gene modified T lymphocytes to a subject for the therapy of disease. Adoptive immunotherapy has yet to realize its potential for treating a wide variety of diseases including cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. However, most, if not all adoptive immunotherapy strategies require T cell activation and expansion steps to generate a clinically effective, therapeutic dose of T cells. Existing strategies of obtaining patient cells, and ex vivo activation, expansion and recovery of effective number of cells for ACT is a prolonged, cumbersome and an inherently complex process—and poses a serious challenge. Accordingly, there remains a need for developing compositions and methods for expansion and induction of antigen specific T cells with a favorable phenotype and function and within a shorter time span.

SUMMARY

Provided herein is a method for treating cancer in a subject in need thereof comprising: selecting at least one epitope sequence from a library of epitope sequences, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele of the subject; and contacting a T cell from the subject or an allogeneic T cell with one or more peptides comprising the at least one selected epitope sequence, wherein each of the at least one selected epitope sequence is pre-validated to satisfy at least three of the following criteria: binds to a protein encoded by an HLA allele of the subject, is immunogenic according to an immunogenicity assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay.

In some embodiments, one or more of the least one selected epitope sequence comprises an epitope that is not expressed by cancer cells of the subject.

In some embodiments, the epitope that is not expressed by cancer cells of the subject is expressed by cells in a tumor microenvironment of the subject.

In some embodiments, an epitope that binds to a protein encoded by an HLA allele of the subject is predicted to bind to an MHC molecule encoded by the HLA allele with an affinity of 500 nM or less using an MHC epitope prediction program implemented on a computer.

In some embodiments, the MHC epitope prediction program implemented on a computer is NetMHCpan In some embodiments, the MHC epitope prediction program implemented on a computer is NetMHCpan version 4.0.

In some embodiments, the epitope that is presented by antigen presenting cells (APCs) according to a mass spectrometry assay are detected by mass spectrometry after elution from the APCs with a mass accuracy of the detected peptide to be less than 15 Da, 10 Da or 5 Da, or less than 10,000 or 5,000 parts per million (ppm).

In some embodiments, the epitope that is immunogenic according to an immunogenicity assay is immunogenic according to a multimer assay or a functional assay.

In some embodiments, the multimer assay comprises flow cytometry analysis.

In some embodiments, the multimer assay comprises detecting T cells bound to a peptide-MHC multimer comprising the at least one selected epitope sequence and the matched HLA allele, wherein the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence.

In some embodiments, epitope is immunogenic according to the multimer assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.1% or 0.01% or 0.005% of the CD8⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ T cells is higher than the percentage of detected T cells of CD8+ T cells detected in a control sample.

In some embodiments, the control sample comprises T cells that have been stimulated with APCs that (i) do not comprise a peptide containing the at least one selected epitope sequence, (ii) comprise a peptide derived from a different protein than the at least one selected epitope sequence, or (iii) comprise a peptide with a random sequence.

In some embodiments, the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 7, 18, 19, 20 or more days.

In some embodiments, antigen-specific T cells have been expanded at least 5-fold, 10-fold, 20, fold, 50-fold, 100-fold, 500-fold or 1,000-fold or more in the presence of APCs comprising a peptide containing the at least one selected epitope sequence.

In some embodiments, the functional assay comprises an immunoassay.

In some embodiments, the functional assay comprises detecting T cells with intracellular staining of IFNγ or TNFα or cell surface expression of CD107a and/or CD107b, wherein the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence

In some embodiments, the epitope is immunogenic according to the functional assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.1% or 0.01% or 0.005% of the CD8⁺ or the CD4⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ or CD4⁺ T cells is higher than the percentage of detected T cells of CD8+ or CD4⁺ T cells detected in a control sample.

In some embodiments, a number of cells presenting the epitope that are killed by the T cells is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 500, or 1,000 fold higher than a number of cells presenting the epitope killed by T cells that have been stimulated with APCs that (i) do not comprise a peptide containing the at least one selected epitope sequence, (ii) comprise a peptide derived from a different protein than the at least one selected epitope sequence, or (iii) comprise a peptide with a random sequence.

In some embodiments, a number of cells presenting a mutant epitope that are killed by the T cells is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 500, or 1,000 fold higher than a number of cells presenting a corresponding wild-type epitope that are killed by the T cells.

In some embodiments, the T cells stimulated to be cytotoxic according to the cytotoxicity assay are T cells stimulated to be specifically cytotoxic according to the cytotoxicity assay.

In some embodiments, the method comprises selecting the subject using a circulating tumor DNA assay.

In some embodiments, the method comprises selecting the subject using a gene panel.

In some embodiments, the T cell is from a biological sample from the subject.

In some embodiments, the T cell is from an apheresis or a leukopheresis sample from the subject.

In some embodiments, the T cell is an allogeneic T cell.

In some embodiments, each of the at least one selected epitope sequence is pre-validated to satisfy each of the following criteria: binds to a protein encoded by an HLA allele of the subject, is immunogenic according to an immunogenicity assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay.

In some embodiments, at least one of the one or more peptides is a synthesized peptide or a peptide expressed from a nucleic acid sequence.

In some embodiments, the method comprises identifying a protein encoded by an HLA allele of the subject or identifying an HLA allele in the genome of the subject.

In some embodiments, the at least one selected epitope sequence is selected from one or more epitope sequences of Table 1A-1F, Table 2A-2C, Table 3, Table 4A-4M, Table 5, Table 6, Table 7, Table 8, Table 11, Table 12, Table 13 and Table 14.

In some embodiments, the method comprises expanding the T cell contacted with the one or more peptides in vitro or ex vivo to obtain a population of T cells specific to the at least one selected epitope sequence in complex with an MEC protein.

In some embodiments, the method further comprises administering the population of T cells to the subject.

In some embodiments, a protein comprising the at least one selected epitope sequence is expressed by a cancer cell of the subject.

In some embodiments, a protein comprising the at least one selected epitope sequences is expressed by cells in the tumor microenvironment of the subject.

In some embodiments, one or more of the at least one selected epitope sequence comprises a mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a tumor specific mutation.

In some embodiments, one or more of the at least one selected epitope sequence is from a protein overexpressed by a cancer cell of the subject.

In some embodiments, one or more of the at least one selected epitope sequence comprises a driver mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a drug resistance mutation.

In some embodiments, one or more of the at least one selected epitope sequence is from a tissue-specific protein.

In some embodiments, one or more of the at least one selected epitope sequence is from a cancer testes protein.

In some embodiments, one or more of the at least one selected epitope sequence is a viral epitope.

In some embodiments, one or more of the at least one selected epitope sequence is a minor histocompatibility epitope.

In some embodiments, one or more of the at least one selected epitope sequence is from a RAS protein.

In some embodiments, one or more of the at least one selected epitope sequence is from a GATA3 protein.

In some embodiments, one or more of the at least one selected epitope sequence is from a EGFR protein.

In some embodiments, one or more of the at least one selected epitope sequence is from a BTK protein.

In some embodiments, one or more of the at least one selected epitope sequence is from a p53 protein.

In some embodiments, one or more of the at least one selected epitope sequence is from aTMPRSS2::ERG fusion polypeptide.

In some embodiments, one or more of the at least one selected epitope sequence is from a Myc protein.

In some embodiments, at least one of the at least one selected epitope sequence is from a protein encoded by a gene selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGES, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNLIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R.

In some embodiments, at least one of the at least one selected epitope sequence is from a tissue-specific protein that has an expression level in a target tissue of the subject that is at least 2 fold more than an expression level of the tissue-specific protein in each tissue of a plurality of non-target tissues that are different than the target tissue.

In some embodiments, contacting a T cell from the subject or an allogeneic T cell with one or more peptides comprising the at least one selected epitope sequence comprises contacting the T cell with APCs presenting the epitope.

In some embodiments, the population of immune cells is from a biological sample from the subject.

In some embodiments, the method further comprises (b) incubating the CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) a polypeptide comprising the at least one selected epitope sequence, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of cells comprising stimulated T cells.

In some embodiments, the method further comprises (c) expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (i) the at least one selected epitope sequence and (ii) an MEC protein expressed by the cancer cells or APCs of the subject.

In some embodiments, the T cells are expanded in less than 28 days.

In some embodiments, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprising tumor antigen specific T cells is at least two-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample.

In some embodiments, at least 0.1% of the CD8+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD8+ tumor antigen-specific T cells derived from naïve CD8+ T cells.

In some embodiments, at least 0.1% of the CD4+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD4+ tumor antigen-specific T cells derived from naïve CD4+ T cells.

In some embodiments, the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs.

In some embodiments, expanding further comprises (C) contacting the expanded population of T cells with a third population of mature APCs, wherein the third population of mature APCs (i) have been incubated with FLT3L and (ii) present the at least one selected epitope sequence; and (D) expanding the expanded population of T cells for a third time period, thereby forming the expanded population of cells comprising tumor antigen-specific T cells.

In some embodiments, the third population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the expanded population of T cells with the third population of mature APCs.

In some embodiments, the biological sample is a peripheral blood sample, a leukapheresis sample or an apheresis sample.

In some embodiments, the method further comprises harvesting the expanded population of cells comprising tumor antigen-specific T cells, cryopreserving the expanded population of cells comprising tumor antigen-specific T cells or preparing a pharmaceutical composition containing the expanded population of cells comprising tumor antigen-specific T cells.

In some embodiments, incubating comprises incubating the CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide.

In some embodiments, the human subject with cancer is the human subject from which the biological sample was obtained.

In some embodiments, the polypeptide is from 8 to 50 amino acids in length.

In some embodiments, the polypeptide comprises at least two of the selected epitope sequence, each expressed by cancer cells of a human subject with cancer.

In some embodiments, depleting further comprising depleting CD19+ cells from the population of immune cells comprising a first population of APCs and T cells.

In some embodiments, depleting further comprising depleting CD11b+ cells from the population of immune cells comprising a first population of APCs and T cells.

In some embodiments, the method comprises generating cancer cell nucleic acids from a first biological sample comprising cancer cells obtained from a subject and generating non-cancer cell nucleic acids from a second biological sample comprising non-cancer cells obtained from the same subject.

In some embodiments, the protein encoded by an HLA allele of the subject is a protein encoded by an HLA allele selected from the group consisting of HLA-A01:01, HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-A24:01, HLA-A30:01, HLA-A31:01, HLA-A32:01, HLA-A33:01, HLA-A68:01, HLA-B07:02, HLA-B08:01, HLA-B15:01, HLA-B44:03, HLA-007:01 and HLA-007:02.

In some embodiments, one or more of the at least one selected epitope sequence has a length of from 8 to 12 amino acids.

In some embodiments, one or more of the at least one selected epitope sequence has a length of from 13-25 amino acids.

In some embodiments, the method comprises isolating genomic DNA or RNA from cancer cells and non-cancer cells of the subject.

In some embodiments, one or more of the at least one selected epitope sequence comprises a point mutation or a sequence encoded by a point mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a neoORF mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a gene fusion mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by an indel mutation.

In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a splice site mutation.

In some embodiments, at least two of the at least one selected epitope sequence are from a same protein.

In some embodiments, at least two of the at least one selected epitope sequence comprise an overlapping sequence.

In some embodiments, at least two of the at least one selected epitope sequence are from different proteins.

In some embodiments, the one or more peptides comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more peptides.

In some embodiments, cancer cells of the subject are cancer cells of a solid cancer.

In some embodiments, cancer cells of the subject are cancer cells of a leukemia or a lymphoma.

In some embodiments, the mutation is a mutation that occur in a plurality of cancer patients.

In some embodiments, the MEC is a Class I MEC.

In some embodiments, the MEC is a Class II MEC.

In some embodiments, the T cell is a CD8 T cell.

In some embodiments, the T cell is a CD4 T cell.

In some embodiments, the T cell is a cytotoxic T cell.

In some embodiments, the T cell is a memory T cell.

In some embodiments, the T cell is a naive T cell.

In some embodiments, the method further comprises selecting one or more subpopulation of cells from an expanded population of T cells prior to administering to the subject.

In some embodiments, eliciting an immune response in the T cell culture comprises inducing IL2 production from the T cell culture upon contact with the peptide.

In some embodiments, eliciting an immune response in the T cell culture comprises inducing a cytokine production from the T cell culture upon contact with the peptide, wherein the cytokine is an Interferon gamma (IFN-γ), Tumor Necrosis Factor (TNF) alpha (α) and/or beta (β) or a combination thereof.

In some embodiments, eliciting an immune response in the T cell culture comprises inducing the T cell culture to kill a cell expressing the peptide.

In some embodiments, eliciting an immune response in the T cell culture comprises detecting an expression of a Fas ligand, granzyme, perforins, IFN, TNF, or a combination thereof in the T cell culture.

In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is purified.

In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is lyophilized.

In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is in a solution.

In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is present in a storage condition such that the integrity of the peptide is ≥99%.

In some embodiments, the method comprises stimulating T cells to be cytotoxic against cells loaded with the at least one selected epitope sequences according to a cytotoxicity assay.

In some embodiments, the method comprises stimulating T cells to be cytotoxic against cancer cells expressing a protein comprising the at least one selected epitope sequences according to a cytotoxicity assay.

In some embodiments, the method comprises stimulating T cells to be cytotoxic against a cancer associated cell expressing a protein comprising the at least one selected epitope sequences according to a cytotoxicity assay.

In some embodiments, the at least one selected epitope is expressed by a cancer cell, and an additional selected epitope is expressed by a cancer associated cell.

In some embodiments, the additional selected epitope is expressed on a cancer associated fibroblast cell.

In some embodiments, the additional selected epitope is selected from Table 8.

Also provided herein is a pharmaceutical composition comprising a T cell produced by a method provided herein.

Also provided herein is a library of polypeptides comprising epitope sequences or polynucleotides encoding the polypeptides, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele; and wherein each epitope sequence in the library is pre-validated to satisfy at least three of the following criteria: binds to a protein encoded by an HLA allele of a subject with cancer to be treated, is immunogenic according to an immunogenic assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and/or stimulates T cells to be cytotoxic according to a cytotoxicity assay.

Also provided herein is a method of treating cancer in a subject comprising administering to the subject (i) a polypeptide comprising a G12R RAS epitope, or (ii) a polynucleotide encoding the polypeptide; wherein: (a) the G12R RAS epitope is vvgaRgvgk (SEQ ID NO: 1) and the subject expresses a protein encoded by an HLA-A03:01 allele; (b) the G12R RAS epitope is eyklvvvgaR (SEQ ID NO: 2) and the subject expresses a protein encoded by an HLA-A33:03 allele; (c) the G12R RAS epitope is vvvgaRgvgk (SEQ ID NO: 3) and the subject expresses a protein encoded by an HLA-A11:01 allele; or (d) the G12R RAS epitope is aRgvgksal (SEQ ID NO: 4) and the subject expresses a protein encoded by an HLA-allele selected from the group consisting of HLA-C07:02, HLA-B39:01 and HLA-C07:01.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is schematic of an exemplary method provided herein to prime, activate and expand antigen-specific T cells.

FIG. 1B is schematic of an exemplary method provided herein to prime, activate and expand antigen-specific T cells.

FIG. 2 is schematic of an exemplary method for offline characterization of shared epitopes.

FIG. 3A depicts data illustrating that in silico epitope prediction identified multiple neoantigens derived from RAS G12D mutations that are presented according to mass spectrometry. Figure discloses SEQ ID NOS 1420, 1421, 1147, 1245, and 1247, respectively, in order of appearance.

FIG. 3B depicts data illustrating that in silico epitope prediction identified multiple neoantigens derived from RAS G12V mutations that are presented according to mass spectrometry. Figure discloses SEQ ID NOS 1422, 1423, 162, 163, and 1148, respectively, in order of appearance.

FIG. 3C depicts data illustrating that in silico epitope prediction identified multiple neoantigens derived from RAS G12C mutations that are presented according to mass spectrometry. Figure discloses SEQ ID NO: 1424.

FIG. 3D depicts data illustrating that in silico epitope prediction identified multiple neoantigens derived from RAS G12R mutations that are presented according to mass spectrometry. Figure discloses SEQ ID NOS 1425, 1426, 1253, and 2, respectively, in order of appearance.

FIG. 4A depicts data illustrating that presentation of shared neoantigen epitopes can be directly confirmed by mass spectrometry and that RAS neoantigens are targetable in defined patient populations.

FIG. 4B shows head-to-toe plot of MS/MS spectra for the endogenously processed mutant RAS peptide epitope VVVGAVGVGK (SEQ ID NO: 5) (top) and its corresponding heavy peptide (bottom). 293T cells were lentivirally transduced with both a polypeptide containing the RAS^G12Vmutant peptide and an HLA-A*03:01 gene.

FIG. 4C shows head-to-toe plot of MS/MS spectra for the endogenously processed mutant RAS peptide epitope VVVGAVGVGK (SEQ ID NO: 5) (top) and its corresponding heavy peptide (bottom). SW620 cells that naturally express the RAS^G12Vmutant were transduced with a lentiviral vector encoding an HLA-A*03:01 gene.

FIG. 4D shows head-to-toe plot of MS/MS spectra for the endogenously processed mutant RAS peptide epitope VVVGAVGVGK (SEQ ID NO: 5) (top) and its corresponding heavy peptide (bottom). NCI-H441 cells naturally expressing both the RAS^G12Vmutation and the HLA-A*03:01 gene were used for this experiment.

FIG. 4E shows head-to-toe plot of MS/MS spectra for the endogenously processed GATA3 neoORF peptide epitope SMLTGPPARV (SEQ ID NO: 6). Endogenous peptide spectrum is shown in the top panel and corresponding light synthetic spectrum is shown in the bottom panels.

FIG. 5 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12 neoantigens on HLA-A11:01 and HLA-A03:01.

FIG. 6 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces multiple de novo CD8 T cell responses against RAS G12V neoantigen on HLA-A11:01. As indicated in the pie charts, the frequency of individual T cell clones induced against RAS G12V neoantigen on HLA-A11:01 in 3 independent healthy donors is depicted.

FIG. 7 depicts data illustrating that RAS^G12V-activated T cells generated ex vivo can kill target cells. A375 target cells expressing GFP were loaded with 2 μM RAS^G12Vantigen, wild-type RAS antigen, or no peptide as control GFP+ cells. RAS^G12V-specific CD8 T cells (effector cells) were incubated with control cells or target cells in a 0.05:1 ratio. In presence of the effector cells, target cells were lysed and depleted more readily that control cells which present either RAS' antigen or no antigen. Graph of specific cell killing as normalized by target cell growth with no peptide is shown in the left diagram. Representative images are shown on the right.

FIG. 8 depicts data illustrating that an exemplary method provided herein to prime, activate and expand RAS G12V-specific T cells with RAS G12V neoantigens on HLA-11:01, but not the corresponding wild-type antigens, induces T cells to become cytotoxic using the indicated effector:target cell ratios and increasing peptide concentration.

FIG. 9 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells with one round (1× stimulated) or two rounds (2× stimulated) of FLT3L-treated PBMCs presenting an epitope with the RAS^G12Vmutation induces T cells to become cytotoxic as measured by AnnexinV positive cells over time after co culturing these T cells with SW620 cells (naturally express the RAS^G12Vmutant) that were transduced with a lentiviral vector encoding an HLA-A*11:01 gene.

FIG. 10 depicts a graph of AnnexinV positive cells over time after co-culturing NCI-H441 cells naturally expressing both the RAS^G12Vmutation and the HLA-A*03:01 gene with T cells that had been primed and activated and expanded with a peptide containing an epitope with the RAS^G12Vmutation at the indicated effector:target cell ratio.

FIG. 11A depicts a graph of IL-2 concentration (pg/mL) vs RAS-G12V wild-type or mutant peptide loaded target cells (A375-A11:01) after incubation in the presence of Jurkat cells transduced with a TCR that binds to the RAS-G12V epitope bound to an MHC encoded by the HLA-A11:01 allele.

FIG. 11B depicts graphs of AnnexinV positive cells over time after co culturing TCR-transduced PBMCs with 5,000 SNGM cells with natural G12V and HLA-A11:01 across a range of effector:target cell ratios.

FIG. 11C depicts a graph of IL-2 concentration (pg/mL) vs RAS-G12V wild-type or mutant peptide loaded target cells (A375-A03:01) after incubation in the presence of Jurkat cells transduced with a TCR that binds to RAS-G12V bound to an MHC encoded by the HLA-A03:01 allele.

FIG. 11D depicts a graph of AnnexinV positive cells over time (top) after co-culturing TCR-transduced PBMCs with cells with natural G12V and HLA-A03:01 using an effector:target cell ratio of 0.75:1 and a graph of IFNγ concentration (pg/mL) after 24 hours of coculturing TCR-transduced PBMCs with cells with natural G12V and HLA-A03:01 using an effector:target cell ratio of 0.75:1.

FIG. 12A depicts a graph of IL-2 concentration (pg/mL) vs FLT3L-treated PBMCs contacted with increasing amounts of the indicated RAS-G12V mutant peptides after being co-cultured with Jurkat cells transduced with a TCR that binds to the underlined RAS-G12V epitope bound to an MHC encoded by the HLA-A11:01 allele. Figure discloses SEQ ID NOS 164, 1427, and 1428, respectively, in order of appearance.

FIG. 12B depicts data illustrating the immunogenicity of the indicated RAS-G12V mutant peptides from FIG. 12A both in vitro using PBMCs from healthy donors (top) and in vivo using HLA-A11:01 transgenic mice immunized with the peptides (bottom).

FIG. 13 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12V neoantigen on HLA-02:01.

FIG. 14 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12 neoantigens on HLA-A68:01.

FIG. 15 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12 neoantigens on HLA-B07:02

FIG. 16 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12 neoantigens on HLA-B08:01.

FIG. 17 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against RAS G12D neoantigen on HLA-008:02.

FIG. 18 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD4 T cell responses against RAS neoantigens.

FIG. 19A depicts data illustrating flow cytometry data demonstrating that enrichment procedures can be used prior to further expansion of antigen-specific T cells. Cells upregulating 4-1BB were enriched using Magnetic-Assisted Cell Separation (MACS; Miltenyi). T cells that were stained by multimers were enriched by MACS on day 14 of stimulation. This approach was able to enrich for multiple antigen-specific T cell populations.

FIG. 19B depicts an exemplary bar graph quantifying the results in FIG. 19A.

FIG. 20 illustrates a summary of experiments illustrating that predicted GATA3 neoORF epitopes have strong affinity (<500 nM), long stability (>0.5 hr) and/or can be detected by mass spectrometry analysis of epitopes eluted from HLA molecules from cells expressing the GATA3 neoORF. Figure discloses SEQ ID NOS 1081, 6, 1088, 1097, 1089, 1085, 1089, 1078, 1093, 1095, 1082, 1079, 1091, 1075, 1078, 1097, 1092, 1079, 1094, and 1096, respectively, in order of appearance.

FIG. 21 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against GATA3 neoORF neoantigens on HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-B07:02 and HLA-B08:01. Figure discloses SEQ ID NOS 1081, 1089, 1089, 1095, and 1091, respectively, in order of appearance.

FIG. 22 depicts data illustrating GATA3 neoORF epitope-activated T cells generated ex vivo can kill target cells. 293T target cells expressing GFP were loaded with 2 μM GATA3 neoORF antigen or left unloaded as control GFP+ cells. GATA3-neoORF-specific CD8 T cells (effector cells) were incubated with control cells or target cells in a 1:10 ratio. In presence of the effector cells, target cells were lysed and depleted more readily that control cells which do present GATA3 neoantigen. Graph of GFP+ cells over 100 hours is shown in the top diagram. Images of the control (bottom left image) and target GFP+ cells (bottom right image) in the presence of GATA3 neoantigen activated CD8 cells are shown.

FIG. 23 depicts a graph of a comparison of Caspase-3 positive fraction of live target cells in GATA3 neoantigen transduced HEK 293T cells versus non-transduced HEK 293T cells. Two different GATA3 induced healthy donor PBMCs were co-cultured with GATA3 neoantigen transduced HEK 293T cells or non-transduced HEK 293T cells as a negative control group.

FIG. 24 depicts flow cytometry data illustrating induction of antigen-specific CD4+ T cells with GATA3 neoORF specific peptide after 20 days in culture, including two stimulations. Antigen-specific T cells are detected by increase in IFNγ and/or TNFα after incubation with GATA3 neoORF peptides (right) relative to no peptides (left)

FIG. 25A depicts a schematic diagram of steps followed through discovery and validation of peptides presented in prostate cancer cell lines or prostate tissue from human donors, and generating validated peptides for a curated validated peptide library.

FIG. 25B depicts data illustrating generation of epitope specific CD8T cells in vitro. The peptides were predicted using T cell epitope prediction software in proteins specific to prostate cancer. Figure discloses SEQ ID NOS 1403, 1405, and 7, respectively, in order of appearance.

FIG. 25C depicts data illustrating KLK4 epitope-activated T cells generated ex vivo are immunogenic and kill target cells. 293T target cells expressing GFP were loaded with 2 μM KLK4 antigen (LLANGRMPTV (SEQ ID NO: 7)) or left unloaded as control GFP+ cells. KLK4 specific CD8 T cells (effector cells) were incubated with control cells or target cells in a 1:10 ratio. In presence of the effector cells, target cell growth was controlled more readily than control cells which do not express KLK4. Also shown is a graph of GFP+ cells over 100 hours (bottom). Images of the control (bottom left image) and target GFP+ cells (bottom right image) in the presence of KLK4 activated CD8 cells are shown.

FIG. 26 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against a BTK C481S neoantigen on HLA-02:01.

FIG. 27 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces de novo CD8 T cell responses against EGFR T790M neoantigens on HLA-02:01.

FIG. 28A depicts a schematic of an exemplary method provided herein for application of T cell therapies.

FIG. 28B depicts a schematic of an exemplary method provided herein for application of T cell therapies.

FIG. 29 depicts a schematic of an exemplary method for in silico T cell epitope prediction. PPV was determined for a given n number of hits and 5,000 decoys, what fraction of the n top-ranked peptides were hits.

FIG. 30 depicts a schematic of allelic coverage of the MHC ligandome using in silico epitope prediction.

FIG. 31 depicts a schematic comparing in silico T cell epitope prediction models.

FIG. 32 depicts a schematic illustrating identification and validation of immunogenic peptides using in silico T cell epitope prediction and an exemplary method provided herein to prime, activate and expand antigen-specific T cells.

FIG. 33 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells can induce and expand multiple neoantigen CD8+ T cell populations. The data shown is representative data from sample from a melanoma patient.

FIG. 34 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells generated three CD4+ populations in the same patient. The data shown is representative data from sample from a melanoma patient.

FIG. 35 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells repeatedly demonstrates T cell inductions across melanoma patient samples.

FIG. 36 depicts representative data from a melanoma patient sample illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces T cells highly specific for mutant epitopes.

FIG. 37 depicts representative data from a melanoma patient sample illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces T cells that are highly functional.

FIG. 38 depicts data illustrating that an exemplary method provided herein to prime, activate and expand antigen-specific T cells induces CD8+ T cells can kill tumor cells.

DETAILED DESCRIPTION

Although many epitopes have the potential to bind to an MEC molecule, few are capable of binding to an MEC molecule when tested experimentally. Although many epitopes also have the potential to potential to be presented by an MEC molecule that can, for example, be detected by mass spectrometry, only a select number of these epitopes can be presented and detected by mass spectrometry. Although many epitopes also have the potential to be immunogenic, when tested experimentally many of these epitopes are not immunogenic, despite being demonstrated to be presented by antigen presenting cells. Many epitopes also have the potential to activate T cells to become cytotoxic; however, many epitopes that have been demonstrated to be presented by antigen presenting cells and/or to be immunogenic are still not capable of activating T cells to become cytotoxic.

Provided herein are antigens containing T cell epitopes that have been identified and validated as binding to one or more MEC molecules, presented by the one or more MEC molecules, being immunogenic and capable of activating T cells to become cytotoxic. The validated antigens and polynucleotides encoding these antigens can be used in preparing antigen specific T cells for therapeutic uses. In some embodiments, the validated antigens and polynucleotides encoding these antigens can be pre-manufactured and stored for use in a method of manufacturing T cells for therapeutic uses. For example, the validated antigens and polynucleotides encoding these antigens can be pre-manufactured or manufactured quickly to prepare therapeutic T cell compositions for patients quickly. Using validated antigens with T cell epitopes, immunogens such as peptides having HLA binding activity or RNA encoding such peptides can be manufactured. Multiple immunogens can be identified, validated and pre-manufactured in a library. In some embodiments, peptides can be manufactured in a scale suitable for storage, archiving and use for pharmacological intervention on a suitable patient at a suitable time.

Some, if not all cancers have antigens that are potential targets for immunotherapy. Each peptide antigen may be presented for T cell activation on an antigen presenting cells in association with a specific HLA-encoded MEC molecule. On the other hand, provided herein is a potentially universal approach, where particular epitopes are pre-identified and pre-validated for particular HLAs, and these epitopes can be pre-manufactured for a cell therapy manufacturing process. For example, a number of KRAS epitopes with G12, G13 and Q61 mutations can be identified using a reliable T cell epitope presentation prediction model (see, e.g., PCT/US2018/017849, filed Feb. 12, 2018, and PCT/US2019/068084 filed Dec. 20, 2019, each of which are incorporated by reference in their entirety), with validation of immunogenicity of these epitopes, processing and presentation using mass spectrometry of these epitopes, and ability to generate cytotoxic T cells with TCRs against these epitopes and MHCs encoded by different HLAs. Each epitope is validated with its specific amino acid sequence and relevant HLA. Once these epitopes are validated, a library can be created containing pre-manufactured immunogens, such as peptides containing the epitopes or RNA encoding peptides containing these epitopes.

The antigens can be non-mutated antigens or mutated antigens. For example, the antigens can be tumor-associated antigens, mutated antigens, tissue-specific antigens or neoantigens. In some embodiments, the antigens are tumor-associated antigens. In some embodiments, the antigens are mutated antigens. In some embodiments, the antigens are tissue-specific antigens. In some embodiments, the antigens are neoantigens. Neoantigens are found in the cancer or the tumor in a subject and is not evident in the germline or expressed in the healthy tissue of the subject. Therefore, for a gene mutation in cancer to satisfy the criteria of generating a neoantigen, the gene mutation in the cancer must be a non-silent mutation that translates into an altered protein product. The altered protein product contains an amino acid sequence with a mutation that can be a mutated epitope for a T cell. The mutated epitope has the potential to bind to an MEC molecule. The mutated epitope also has the potential to be presented by an MEC molecule that can, for example, be detected by mass spectrometry. Furthermore, the mutated epitope has the potential to be immunogenic. Additionally, the mutated epitope has the potential to activate T cells to become cytotoxic.

Provided herein is a method for treating cancer in a subject in need thereof comprising selecting at least one epitope sequence from a library of epitope sequences, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele of the subject; and contacting a T cell from the subject or an allogeneic T cell with one or more peptides comprising the at least one selected epitope sequence, wherein each of the at least one selected epitope sequence is pre-validated to satisfy at least two or three or four of the following criteria binds to a protein encoded by an HLA allele of the subject, is immunogenic according to an immunogenicity assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay. In some embodiments, the method further comprises administering the population of T cells to the subject.

In some embodiments, the at least one selected epitope sequence comprises a mutation and the method comprises identifying cancer cells of the subject to encode the epitope with the mutation; the at least one selected epitope sequence is within a protein overexpressed by cancer cells of the subject and the method comprises identifying cancer cells of the subject to overexpress the protein containing the epitope; or the at least one epitope sequence comprises a protein expressed by a cell in a tumor microenvironment. In some embodiments, one or more of the least one selected epitope sequence comprises an epitope that is not expressed by cancer cells of the subject. In some embodiments, the epitope that is not expressed by cancer cells of the subject is expressed by cells in a tumor microenvironment of the subject. In some embodiments, the method comprises selecting the subject using a circulating tumor DNA assay. In some embodiments, the method comprises selecting the subject using a gene panel.

In some embodiments, the T cell is from a biological sample from the subject. In some embodiments, the T cell is from an apheresis or a leukopheresis sample from the subject. In some embodiments, the T cell is an allogeneic T cell.

In some embodiments, each of the at least one selected epitope sequence is pre-validated to satisfy one or more or each of the following criteria: binds to a protein encoded by an HLA allele of the subject, is immunogenic according to an immunogenicity assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay.

In some embodiments, an epitope that binds to a protein encoded by an HLA allele of the subject binds to an MHC molecule encoded by the HLA allele with an affinity of 500 nM or less according to a binding assay. For example, an epitope that binds to a protein encoded by an HLA allele of the subject can bind to an MHC molecule encoded by the HLA allele with an affinity of 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 75 nM, 50 nM, or 25 nM or less according to a binding assay. In some embodiments, an epitope that binds to a protein encoded by an HLA allele of the subject is predicted to bind to an MHC molecule encoded by the HLA allele with an affinity of 500 nM or less using an MHC epitope prediction program implemented on a computer. For example, an epitope that binds to a protein encoded by an HLA allele of the subject can be predicted to bind to an MHC molecule encoded by the HLA allele with an affinity of 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 75 nM, 50 nM, or 25 nM or less using an MHC epitope prediction program implemented on a computer. In some embodiments, the MHC epitope prediction program implemented on a computer is NetMHCpan. In some embodiments, the MHC epitope prediction program implemented on a computer is NetMHCpan version 4.0.

In some embodiments, the epitope that is presented by antigen presenting cells (APCs) according to a mass spectrometry assay is detected by mass spectrometry after elution from the APCs with a mass accuracy of the detected peptide to be less than 15 Da. For example, the epitope that is presented by antigen presenting cells (APCs) according to a mass spectrometry assay can be detected by mass spectrometry after elution from the APCs with a mass accuracy of the detected peptide to be less than 14 Da, 13 Da, 12 Da, 11 Da, 10 Da, 9 Da, 8 Da, 7 Da, 6 Da, 5 Da, 4 Da, 3 Da, 2 Da, or 1 Da. In some embodiments, the epitope that is presented by antigen presenting cells (APCs) according to a mass spectrometry assay is detected by mass spectrometry after elution from the APCs with a mass accuracy of the detected peptide to be less than 10,000 parts per million (ppm). For example, the epitope that is presented by antigen presenting cells (APCs) according to a mass spectrometry assay can be detected by mass spectrometry after elution from the APCs with a mass accuracy of the detected peptide to be less than 7,500 ppm; 5,000 ppm; 2,500 ppm; 1,000 ppm; 900 ppm; 800 ppm; 700 ppm; 600 ppm; 500 ppm; 400 ppm; 300 ppm; 200 ppm or 100 ppm.

In some embodiments, the epitope that is immunogenic according to an immunogenicity assay is immunogenic according to a multimer assay. In some embodiments, the multimer assay comprises flow cytometry analysis. In some embodiments, the multimer assay comprises detecting T cells bound to a peptide-MHC multimer comprising the at least one selected epitope sequence and the matched HLA allele, wherein the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence. In some embodiments, an epitope is immunogenic according to the multimer assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.005% of the CD8⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ T cells is higher than the percentage of detected T cells of CD8+ T cells detected in a control sample. For example, an epitope can be immunogenic according to the multimer assay when (i) at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.005% of the CD8+ cells analyzed, and (iii) the percentage of detected T cells of CD8+ T cells is higher than the percentage of detected T cells of CD8+ T cells detected in a control sample. For example, an epitope can be immunogenic according to the multimer assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ T cells is higher than the percentage of detected T cells of CD8+ T cells detected in a control sample. For example, an epitope can be immunogenic according to the multimer assay when (i) at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8+ cells analyzed, and (iii) the percentage of detected T cells of CD8+ T cells is higher than the percentage of detected T cells of CD8+ T cells detected in a control sample.

In some embodiments, the epitope is immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least one out of six stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least one out of six stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2 out of 6, 7, 8, 9, 10, 11 or 12 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5 or 6 out of 6 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6 or 7 out of 7 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6, 7 or 8 out of 8 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6, 7, 8 or 9 out of 9 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 out of 10 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 out of 11 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 out of 12 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 3 out of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 4 out of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least one out of six stimulations from the same starting sample. For example, the epitope can be immunogenic according to the multimer assay when at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected in at least 2 out of 6, 7, 8, 9, 10, 11 or 12 stimulations from the same starting sample or in at least 3 out of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 stimulations from the same starting sample or in at least 4 out of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 stimulations from the same starting sample. In some embodiments, the control sample comprises T cells that have been stimulated with APCs that (i) do not comprise a peptide containing the at least one selected epitope sequence, (ii) comprise a peptide derived from a different protein than the at least one selected epitope sequence, or (iii) comprise a peptide with a random sequence. In some embodiments, the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 7, 18, 19, 20 or more days. In some embodiments, antigen-specific T cells have been expanded at least 5-fold, 10-fold, 20, fold, 50-fold, 100-fold, 500-fold or 1,000-fold or more in the presence of APCs comprising a peptide containing the at least one selected epitope sequence.

In some embodiments, the epitope that is immunogenic according to an immunogenicity assay is immunogenic according to a functional assay. In some embodiments, the functional assay comprises an immunoassay. In some embodiments, the functional assay comprises detecting T cells with intracellular staining of IFNγ or TNFα or cell surface expression of CD107a and/or CD107b, wherein the T cells have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence In some embodiments, the epitope is immunogenic according to the functional assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.005% of the CD8⁺ or the CD4⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ or CD4⁺ T cells is higher than the percentage of detected T cells of CD8+ or CD4⁺ T cells detected in a control sample. For example the epitope can be immunogenic according to the functional assay when (i) at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.005% of the CD8⁺ or the CD4⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ or CD4⁺ T cells is higher than the percentage of detected T cells of CD8+ or CD4⁺ T cells detected in a control sample. For example the epitope can be immunogenic according to the functional assay when (i) at least 10 T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8⁺ or the CD4⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ or CD4⁺ T cells is higher than the percentage of detected T cells of CD8+ or CD4⁺ T cells detected in a control sample. For example the epitope can be immunogenic according to the functional assay when (i) at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or more T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence are detected, (ii) the detected T cells make up at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8⁺ or the CD4⁺ cells analyzed, and (iii) the percentage of detected T cells of CD8+ or CD4⁺ T cells is higher than the percentage of detected T cells of CD8+ or CD4⁺ T cells detected in a control sample.

In some embodiments, the T cells stimulated to be cytotoxic according to the cytotoxicity assay are T cells that have been stimulated with APCs comprising a peptide containing the at least one selected epitope sequence that kill cells presenting the epitope. In some embodiments, a number of cells presenting the epitope that are killed by the T cells is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 500, or 1,000 fold higher than a number of cells that do not present the epitope that are killed by the T cells. In some embodiments, a number of cells presenting the epitope that are killed by the T cells is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 500, or 1,000 fold higher than a number of cells presenting the epitope killed by T cells that have been stimulated with APCs that (i) do not comprise a peptide containing the at least one selected epitope sequence, (ii) comprise a peptide derived from a different protein than the at least one selected epitope sequence, or (iii) comprise a peptide with a random sequence In some embodiments, a number of cells presenting a mutant epitope that are killed by the T cells is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 100, 500, or 1,000 fold higher than a number of cells presenting a corresponding wild-type epitope that are killed by the T cells. In some embodiments, the T cells stimulated to be cytotoxic according to the cytotoxicity assay are T cells stimulated to be specifically cytotoxic according to the cytotoxicity assay.

In some embodiments, at least one of the one or more peptides is a synthesized peptide or a peptide expressed from a nucleic acid sequence.

In some embodiments, the method comprises identifying a protein encoded by an HLA allele of the subject or identifying an HLA allele in the genome of the subject.

In some embodiments, the at least one selected epitope sequence is selected from one or more epitope sequences of Table 1-8 and 11-14.

In some embodiments, a protein comprising the at least one selected epitope sequence is expressed by a cancer cell of the subject. In some embodiments, a protein comprising the at least one selected epitope sequences is expressed by cells in the tumor microenvironment of the subject.

In some embodiments, one or more of the at least one selected epitope sequence comprises a mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a tumor specific mutation. In some embodiments, one or more of the at least one selected epitope sequence is from a protein overexpressed by a cancer cell of the subject. In some embodiments, one or more of the at least one selected epitope sequence comprises a driver mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a drug resistance mutation. In some embodiments, one or more of the at least one selected epitope sequence is from a tissue-specific protein. In some embodiments, one or more of the at least one selected epitope sequence is from a cancer testes protein. In some embodiments, one or more of the at least one selected epitope sequence is a viral epitope. In some embodiments, one or more of the at least one selected epitope sequence is a minor histocompatibility epitope. In some embodiments, one or more of the at least one selected epitope sequence is from a RAS protein. In some embodiments, one or more of the at least one selected epitope sequence is from a GATA3 protein. In some embodiments, one or more of the at least one selected epitope sequence is from a EGFR protein. In some embodiments, one or more of the at least one selected epitope sequence is from a BTK protein. In some embodiments, one or more of the at least one selected epitope sequence is from a p53 protein. In some embodiments, one or more of the at least one selected epitope sequence is from aTMPRSS2::ERG fusion polypeptide. In some embodiments, one or more of the at least one selected epitope sequence is from a Myc protein. In some embodiments, at least one of the at least one selected epitope sequence is from a protein encoded by a gene selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGES, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNLIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, IAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R.

In some embodiments, the APCs presenting the epitope comprises one or more peptides comprising the at least one selected epitope sequence or a polynucleic acid that encodes one or more peptides comprising the at least one selected epitope sequence. In some embodiments, the polypeptide comprises at least two of the selected epitope sequence, each expressed by cancer cells of a human subject with cancer.

In some embodiments, the method comprises depleting CD14+ cells and CD25+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells. In some embodiments, the population of immune cells is from a biological sample from the subject. In some embodiments, the method further comprises incubating the CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and a polypeptide comprising the at least one selected epitope sequence, or a polynucleotide encoding the polypeptide; thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method further comprises expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising the at least one selected epitope sequence and an MHC protein expressed by the cancer cells or APCs of the subject. In some embodiments, expanding is performed in less than 28 days. In some embodiments, incubating comprises incubating the CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide. In some embodiments, depleting CD14+ cells and CD25+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprising a first population of APCs and T cells with a CD14 binding agent and a CD25 binding agent. In some embodiments, depleting further comprising depleting CD19+ cells from the population of immune cells comprising a first population of APCs and T cells. In some embodiments, depleting further comprising depleting CD11b+ cells from the population of immune cells comprising a first population of APCs and T cells.

In some embodiments, the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen specific T cells is at least two-fold higher than the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample. In some embodiments, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprising tumor antigen specific T cells is at least two-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample. In some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD8+ tumor antigen-specific T cells derived from naïve CD8+ T cells. In some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD8+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD8+ tumor antigen-specific T cells derived from memory CD8+ T cells. In some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD4+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD4+ tumor antigen-specific T cells derived from naïve CD4+ T cells. In some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the CD4+ T cells in the expanded population of cells comprising tumor antigen specific T cells are CD4+ tumor antigen-specific T cells derived from memory CD4+ T cells.

In some embodiments, expanding comprises contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs have been incubated with FLT3L and present the at least one selected epitope sequence; and expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some embodiments, the second population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, expanding further comprises contacting the expanded population of T cells with a third population of mature APCs, wherein the third population of mature APCs have been incubated with FLT3L and present the at least one selected epitope sequence; and expanding the expanded population of T cells for a third time period, thereby forming the expanded population of cells comprising tumor antigen-specific T cells. In some embodiments, the third population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the expanded population of T cells with the third population of mature APCs. In some embodiments, the biological sample is a peripheral blood sample, a leukapheresis sample or an apheresis sample.

In some embodiments, the method comprises identifying one or two or more different proteins that comprise the at least one selected epitope sequence and that are expressed by cancer cells of the subject. In some embodiments, the method comprises identifying one or two or more different proteins that comprise the at least one selected epitope sequence and that are expressed by cancer cells of the subject by measuring levels of RNA encoding the one or two or more different proteins in the cancer cells. In some embodiments, the method comprises isolating genomic DNA or RNA from cancer cells and non-cancer cells of the subject.

In some embodiments, one or more of the at least one selected epitope sequence comprises a point mutation or a sequence encoded by a point mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a neoORF mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a gene fusion mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by an indel mutation. In some embodiments, one or more of the at least one selected epitope sequence comprises a sequence encoded by a splice site mutation. In some embodiments, at least two of the at least one selected epitope sequence are from a same protein. In some embodiments, at least two of the at least one selected epitope sequence comprise an overlapping sequence. In some embodiments, at least two of the at least one selected epitope sequence are from different proteins. In some embodiments, the one or more peptides comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more peptides.

In some embodiments, cancer cells of the subject are cancer cells of a solid cancer. In some embodiments, cancer cells of the subject are cancer cells of a leukemia or a lymphoma.

In some embodiments, the mutation is a mutation that occurs in a plurality of cancer patients.

In some embodiments, the MEC is a Class I MEC. In some embodiments, the MHC is a Class II MHC.

In some embodiments, the T cell is a CD8 T cell. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell t is a memory T cell. In some embodiments, the T cell is a naive T cell.

In some embodiments, the method further comprises selecting one or more subpopulation of cells from an expanded population of T cells prior to administering to the subject.

In some embodiments, eliciting an elicit an immune response in the T cell culture comprises inducing IL2 production from the T cell culture upon contact with the peptide. In some embodiments, eliciting an immune response in the T cell culture comprises inducing a cytokine production from the T cell culture upon contact with the peptide, wherein the cytokine is an Interferon gamma (IFN-γ), Tumor Necrosis Factor (TNF) alpha (α) and/or beta (β) or a combination thereof. In some embodiments, eliciting an immune response in the T cell culture comprises inducing the T cell culture to kill a cell expressing the peptide. In some embodiments, eliciting an immune response in the T cell culture comprises detecting an expression of a Fas ligand, granzyme, perforins, IFN, TNF, or a combination thereof in the T cell culture.

In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is purified. In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is lyophilized. In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is in a solution. In some embodiments, the one or more peptides comprising the at least one selected epitope sequence is present in a storage condition such that the integrity of the peptide is ≥99%.

In some embodiments, the method comprises stimulating T cells to be cytotoxic against cells loaded with the at least one selected epitope sequences according to a cytotoxicity assay. In some embodiments, the method comprises stimulating T cells to be cytotoxic against cancer cells expressing a protein comprising the at least one selected epitope sequences according to a cytotoxicity assay. In some embodiments, the method comprises stimulating T cells to be cytotoxic against a cancer associated cell expressing a protein comprising the at least one selected epitope sequences according to a cytotoxicity assay.

In some embodiments, the at least one selected epitope is expressed by a cancer cell, and an additional selected epitope is expressed by a cancer associated cell. In some embodiments, the additional selected epitope is expressed on a cancer associated fibroblast cell. In some embodiments, the additional selected epitope is selected from Table 8.

In some embodiments, a method provided herein is a method for treating cancer in a subject in need thereof comprising: selecting at least one epitope sequence from a library of epitope sequences, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele; and contacting a T cell from the subject or an allogeneic T cell with one or more peptides comprising the at least one selected epitope sequence, wherein each of the at least one selected epitope sequences; binds to a protein encoded by an HLA allele of the subject; is immunogenic according to an immunogenic assay; is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay.

In some embodiments, the method comprises selecting the subject using a circulating tumor DNA assay. In some embodiments, the method comprises selecting the subject using a gene panel.

In some embodiments, the T cell is from a biological sample from the subject. In some embodiments, the T cell is from an apheresis or a leukopheresis sample from the subject.

In some embodiments, at least one of the one or more peptides a synthesized peptide or a peptide expressed from a nucleic acid sequence.

In some embodiments, the method comprises identifying a protein encoded by an HLA allele of the subject or identifying an HLA allele in the genome of the subject. In some embodiments, the method comprises identifying a protein encoded by an HLA allele of the subject that is expressed by the subject. In some embodiments, the method comprises contacting a T cell from the subject with one or more peptides selected from one or more peptides of a table provided herein. In some embodiments, the method comprises contacting a T cell from the subject with one or more peptides comprising an epitope selected from an epitope of a table provided herein. In some embodiments, the method further comprises expanding in vitro or ex vivo the T cell contacted with the one or more peptides to obtain a population of T cells. In some embodiments, the method further comprises administering the population of T cells to the subject at a dose and a time interval such that the cancer is reduced or eliminated.

In some embodiments, at least one of the one or more peptides is expressed by a cancer cell of the subject. In some embodiments, at least one of the epitopes of the one or more peptides comprises a mutation.

In some embodiments, at least one of the epitopes of the one or more peptides comprises a tumor specific mutation. In some embodiments, at least one of the epitopes of the one or more peptides is from a protein overexpressed by a cancer cell of the subject. In some embodiments, at least one of the epitopes of the one or more peptides is from a protein encoded by a gene selected from the group consisting of ANKRD30A, COL10A1, CTCFL, PPIAL4G, POTEE, DLL3, MMP13, SSX1, DCAF4L2, MAGEA4, MAGEA11, MAGEC2, MAGEA12, PRAME, CLDN6, EPYC, KLK3, KLK2, KLK4, TGM4, POTEG, RLN1, POTEH, SLC45A2, TSPAN10, PAGES, CSAG1, PRDM7, TG, TSHR, RSPH6A, SCXB, HIST1H4K, ALPPL2, PRM2, PRM1, TNP1, LELP1, HMGB4, AKAP4, CETN1, UBQLN3, ACTL7A, ACTL9, ACTRT2, PGK2, C2orf53, KIF2B, ADAD1, SPATA8, CCDC70, TPD52L3, ACTL7B, DMRTB1, SYCN, CELA2A, CELA2B, PNLIPRP1, CTRC, AMY2A, SERPINI2, RBPJL, AQP12A, LAPP, KIRREL2, G6PC2, AQP12B, CYP11B1, CYP11B2, STAR, CYP11A1, and MC2R.

In some embodiments, at least one of the one or more peptides is from a protein encoded by a tissue-specific antigen epitope gene that has an expression level in a target tissue of the subject that is at least 2 fold more than an expression level of the tissue-specific antigen gene in each tissue of a plurality of non-target tissues that are different than the target tissue.

In some embodiments, the method comprises: incubating one or more antigen presenting cell (APC) preparations with a population of immune cells from a biological sample depleted of cells expressing CD14 and CD25 for one or more separate time periods; incubating one or more APC preparations with a population of immune cells from a biological sample for one or more separate time periods, wherein the one or more APCs comprise one or more FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs; or incubating FLT3L and at least one peptide with a population of immune cells from a biological sample, wherein the FLT3L is incubated with the population of immune cells for a first time period and wherein the at least one peptide is incubated with the population of immune cells for a first peptide stimulation time period, thereby obtaining a first stimulated T cell sample, wherein the population of immune cells comprises at least one T cell and at least one APC; wherein at least one antigen specific memory T cell is expanded, or at least one antigen specific naïve T cell is induced.

In some embodiments, the method comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods of less than 28 days from incubating the population of immune cells with a first APC preparation of the one or more APC preparations. In some embodiments, the method comprises incubating a population of immune cells from a biological sample with 3 or less APC preparations for 3 or less separate time periods. In some embodiments, the method comprises incubating a population of immune cells from a biological sample with 2 or less APC preparations for 2 or less separate time periods. In some embodiments, the method comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods of less than 28 days from incubating the population of immune cells with a first APC preparation of the one or more APC preparations. In some embodiments, the total period of preparation of T cells stimulated with an antigen by incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods is less than 28 days.

In some embodiments, at least two of the one or more APC preparations comprise a FLT3L-stimulated APC. In some embodiments, at least three of the one or more APC preparations comprise a FLT3L-stimulated APC. In some embodiments, incubating comprises incubating a first APC preparation of the APC preparations to the T cells for more than 7 days. In some embodiments, an APC of the APC preparations comprises an APC loaded with one or more antigen peptides comprising one or more of the at least one antigen peptide sequence. In some embodiments, an APC of the APC preparations is an autologous APC or an allogenic APC. In some embodiments, an APC of the APC preparations comprises a dendritic cell (DC). In some embodiments, the DC is a CD141⁺ DC. In some embodiments, the method comprises depleting cells expressing CD14 and CD25 from the biological sample, thereby obtaining the population of immune cells from a biological sample depleted of cells expressing CD14 and CD25. In some embodiments, the method further comprises depleting cells expressing CD19. In some embodiments, the method further comprises depleting cells expressing CD11b. In some embodiments, depleting cells expressing CD14 and CD25 comprises binding a CD14 or CD25 binding agent to an APC of the one or more APC preparations. In some embodiments, the method further comprises administering one or more of the at least one antigen specific T cell to a subject.

In some embodiments, incubating comprises incubating a first APC preparation of the one or more APC preparations to the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises incubating at least one of the one or more of the APC preparations with a first medium comprising at least one cytokine or growth factor for a first time period. In some embodiments, the method comprises incubating at least one of the one or more of the APC preparations with a second medium comprising one or more cytokines or growth factors for a third time period, thereby obtaining a matured APC. In some embodiments, the method further comprises removing the one or more cytokines or growth factors of the second medium after the third time period. In some embodiments, an APC of the APC preparations is stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof.

In some embodiments, the antigen is a neoantigen, a tumor associated antigen, a viral antigen, a minor histocompatibility antigen or a combination thereof.

In some embodiments, the method is performed ex vivo.

In some embodiments, wherein the method comprises incubating the population of immune cells from a biological sample depleted of cells expressing CD14 and CD25 with FLT3L for a first time period. In some embodiments, the method comprises incubating at least one peptide with the population of immune cells from a biological sample depleted of cells expressing CD14 and CD25 for a second time period, thereby obtaining a first matured APC peptide loaded sample. In some embodiments, the method comprises depleting cells expressing CD14, cells expressing CD19 and cells expressing CD25 from the population of immune cells. In some embodiments, the method comprises depleting cells expressing CD14, cells expressing CD11b and cells expressing CD25 from the population of immune cells. In some embodiments, the method comprises depleting cells expressing CD14, cells expressing CD11b, cells expressing CD19 and cells expressing CD25. In some embodiments, the method comprises depleting at least CD14, CD11b, CD19 and CD25. In some embodiments, the method comprises depleting cells expressing at least one of CD14, CD11b, CD19 and CD25, and at least a fifth cell type expressing a fifth cell surface marker. In some embodiments, the method comprises selectively depleting CD14 and CD25 expressing cells from the population of immune cells, and any one or more of CD19, CD11b expressing cells, from the population of immune cells, at a first incubation period, at a second incubation period, and/or at a third incubation period.

In some embodiments of the method described herein, contacting a T cell from the subject or an allogeneic T cell with one or more peptides comprising the at least one selected epitope sequence comprises contacting the T cell with APCs presenting the epitope.

In some embodiments of the method described herein, the APCs presenting the epitope comprises one or more peptides comprising the at least one selected epitope sequence or a polynucleic acid that encodes one or more peptides comprising the at least one selected epitope sequence.

In some embodiments of the method described herein, expanding comprises contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs have been incubated with FLT3L and present the at least one selected epitope sequence and expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some embodiments, the second population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the expanding further comprises contacting the expanded population of T cells with a third population of mature APCs, wherein the third population of mature APCs have been incubated with FLT3L and present the at least one selected epitope sequence; and expanding the expanded population of T cells for a third time period, thereby forming the expanded population of cells comprising tumor antigen-specific T cells. In some embodiments, the third population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the expanded population of T cells with the third population of mature APCs. In some embodiments of the method described herein, the method further comprises harvesting the expanded population of cells comprising tumor antigen-specific T cells, cryopreserving the expanded population of cells comprising tumor antigen-specific T cells or preparing a pharmaceutical composition containing the expanded population of cells comprising tumor antigen-specific T cells. In some embodiments, the incubating comprises incubating the CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide.

In some embodiments, the method further comprises administering a pharmaceutical composition comprising the expanded population of cells comprising tumor antigen specific T cells to a human subject with cancer. In some embodiments, the human subject with cancer is the human subject from which the biological sample was obtained. In some embodiments, the polypeptide is from 8 to 50 amino acids in length. In some embodiments, the polypeptide comprises at least two of the selected epitope sequence, each expressed by cancer cells of a human subject with cancer.

In some embodiments, depleting CD14+ cells and CD25+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprising a first population of APCs and T cells with a CD14 binding agent and a CD25 binding agent. In some embodiments, depleting further comprising depleting CD19+ cells from the population of immune cells comprising a first population of APCs and T cells. In some embodiments, the method further comprises contacting the population of immune cells with a CD19 binding agent. In some embodiments, depleting further comprising depleting CD11b+ cells from the population of immune cells comprising a first population of APCs and T cells. In some embodiments, the method further comprises contacting the population of immune cells with a CD11b binding agent.

In some embodiments, the method comprises incubating the first matured APC peptide loaded sample with at least one T cell for a third time period, thereby obtaining a stimulated T cell sample. In some embodiments, the method comprises incubating a T cell of a first stimulated T cell sample with a FLT3L-stimulated APC of a matured APC sample for a fourth time period, FLT3L and a second APC peptide loaded sample of a matured APC sample for a fourth time period or FLT3L and a FLT3L-stimulated APC of a matured APC sample for a fourth time period, thereby obtaining a stimulated T cell sample. In some embodiments, the method comprises incubating a T cell of a second stimulated T cell sample with a FLT3L-stimulated APC of a matured APC sample for a fifth time period, FLT3L and a third APC peptide loaded sample of a matured APC sample for a fifth time period, or FLT3L and a third APC peptide loaded sample of a matured APC sample for a fifth time period, thereby obtaining a stimulated T cell sample.

In some embodiments, the one or more separate time periods, the 3 or less separate time periods, the first time period, the second time period, the third time period, the fourth time period, or the fifth time period is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 31 hours, at least 32 hours, at least 33 hours, at least 34 hours, at least 35 hours, at least 36 hours, at least 37 hours, at least 38 hours, at least 39 hours, or at least 40 hours.

In some embodiments, the one or more separate time periods, the 3 or less separate time periods, the first time period, the second time period, the third time period, the fourth time period, or the fifth time period is from 1 to 4 hours, from 1 to 3 hours, from 1 to 2 hours, from 4 to 40 hours, from 7 to 40 hours, from 4 to 35 hours, from 4 to 32 hours, from 7 to 35 hours or from 7 to 32 hours.

In some embodiments, the population of immune cells comprises the APC or at least one of the one or more APC preparations. In some embodiments, the population of immune cells does not comprise the APC and/or the population of immune cells does not comprise one of the one or more APC preparations.

In some embodiments, the method comprises incubating FLT3L and at least one peptide with a population of immune cells from a biological sample, wherein the FLT3L is incubated with the population of immune cells for a first time period and wherein the at least one peptide is incubated with the population of immune cells for a first peptide stimulation time period, thereby obtaining a first stimulated T cell sample, wherein the population of immune cells comprises at least one T cell and at least one APC. In some embodiments, the method comprises incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a second time period and wherein the at least one peptide is incubated with the at least one APC for a second peptide stimulation time period, thereby obtaining a first matured APC peptide loaded sample; and incubating the first matured APC peptide loaded sample with the first stimulated T cell sample, thereby obtaining a second stimulated T cell sample. In some embodiments, the method comprises incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a third time period and wherein the at least one peptide is incubated with the at least one APC for a third peptide stimulation time period, thereby obtaining a second matured APC peptide loaded sample; and incubating the second matured APC peptide loaded sample with the second stimulated T cell sample, thereby obtaining a third stimulated T cell sample.

In some embodiments, the method further comprises isolating the first stimulated T cell from the stimulated T cell sample. In some embodiments, isolating as described in the preceding sentence comprises enriching a stimulated T cell from a population of immune cells that have been contacted with the at least one APC incubated with the at least one peptide. In some embodiments, the enriching comprises determining expression of one or more cell markers of at least one the stimulated T cell and isolating the stimulated T cell expressing the one or more cell markers. In some embodiments the cell surface markers may be but not limited to one or more of TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cell markers comprise a cytokine.

In some embodiments, the method comprises administering at least one T cell of a first or a second or a third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises: obtaining a biological sample from a subject comprising at least one antigen presenting cell (APC); enriching cells expressing CD14 from the biological sample, thereby obtaining a CD14⁺ cell enriched sample; incubating the CD14⁺ cell enriched sample with at least one cytokine or growth factor for a first time period; incubating at least one peptide with the CD14⁺ cell enriched sample of for a second time period, thereby obtaining an APC peptide loaded sample; incubating the APC peptide loaded sample with one or more cytokines or growth factors for a third time period, thereby obtaining a matured APC sample; incubating APCs of the matured APC sample with a CD14 and CD25 depleted sample comprising T cells for a fourth time period; incubating the T cells with APCs of a matured APC sample for a fifth time period; incubating the T cells with APCs of a matured APC sample for a sixth time period; and administering at least one T cell of the T cells to a subject in need thereof.

In some embodiments, the method comprises: obtaining a biological sample from a subject comprising at least one APC and at least one T cell; depleting cells expressing CD14 and CD25 from the biological sample, thereby obtaining a CD14 and CD25 cell depleted sample; incubating the CD14 and CD25 cell depleted sample with FLT3L for a first time period; incubating at least one peptide with the CD14 and CD25 cell depleted sample of for a second time period, thereby obtaining an APC peptide loaded sample; incubating the APC peptide loaded sample with the at least one T cell for a third time period, thereby obtaining a first stimulated T cell sample; incubating a T cell of the first stimulated T cell sample with an APC of a matured APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; optionally, incubating a T cell of the second stimulated T cell sample with an APC of a matured APC sample for a fifth time period, thereby obtaining a third stimulated T cell sample; administering at least one T cell of the first, the second or the third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises: obtaining a biological sample from a subject comprising at least one APC and at least one T cell; depleting cells expressing CD14 and CD25 from the biological sample, thereby obtaining a CD14 and CD25 cell depleted sample; incubating the CD14 and CD25 cell depleted sample with FLT3L for a first time period; incubating at least one peptide with the CD14 and CD25 cell depleted sample of for a second time period, thereby obtaining an APC peptide loaded sample; incubating the APC peptide loaded sample with the at least one T cell for a third time period, thereby obtaining a first stimulated T cell sample; optionally, incubating a T cell of the first stimulated T cell sample with a FLT3L-stimulated APC of a matured APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; optionally, incubating a T cell of the second stimulated T cell sample with a FLT3L-stimulated APC of a matured APC sample for a fifth time period, thereby obtaining a third stimulated T cell sample; administering at least one T cell of the first, the second or the third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises: obtaining a biological sample from a subject comprising at least one APC and at least one T cell; depleting cells expressing CD14 and CD25 from the biological sample, thereby obtaining a CD14 and CD25 cell depleted sample; incubating the CD14 and CD25 cell depleted sample with FLT3L for a first time period; incubating at least one peptide with the CD14 and CD25 cell depleted sample of for a second time period, thereby obtaining a first APC peptide loaded sample; incubating the first APC peptide loaded sample with the at least one T cell for a third time period, thereby obtaining a first stimulated T cell sample; optionally, incubating a T cell of the first stimulated T cell sample with FLT3L and a second APC peptide loaded sample of a matured APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; optionally, incubating a T cell of the second stimulated T cell sample with FLT3L and a third APC peptide loaded sample of a matured APC sample for a fifth time period, thereby obtaining a third stimulated T cell sample; administering at least one T cell of the first, the second or the third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises: obtaining a biological sample from a subject comprising at least one APC and at least one T cell; depleting cells expressing CD14 and CD25 from the biological sample, thereby obtaining a CD14 and CD25 cell depleted sample; incubating the CD14 and CD25 cell depleted sample with FLT3L for a first time period; incubating at least one peptide with the CD14 and CD25 cell depleted sample of for a second time period, thereby obtaining a first APC peptide loaded sample; incubating the first APC peptide loaded sample with the at least one T cell for a third time period, thereby obtaining a first stimulated T cell sample; optionally, incubating a T cell of the first stimulated T cell sample with FLT3L and a FLT3L-stimulated APC of a matured APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; optionally, incubating a T cell of the second stimulated T cell sample with FLT3L and a FLT3L-stimulated APC of a matured APC sample for a fifth time period, thereby obtaining a third stimulated T cell sample; administering at least one T cell of the first, the second or the third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises: incubating FLT3L and at least one peptide with a population of immune cells from a biological sample, wherein the FLT3L is incubated with the population of immune cells for a first time period and wherein the at least one peptide is incubated with the population of immune cells for a first peptide stimulation time period, thereby obtaining a first stimulated T cell sample, wherein the population of immune cells comprises at least one T cell and at least one APC; optionally, incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a second time period and wherein the at least one peptide is incubated with the at least one APC for a second peptide stimulation time period, thereby obtaining a first matured APC peptide loaded sample; and incubating the first matured APC peptide loaded sample with the first stimulated T cell sample, thereby obtaining a second stimulated T cell sample; optionally, incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a third time period and wherein the at least one peptide is incubated with the at least one APC for a third peptide stimulation time period, thereby obtaining a second matured APC peptide loaded sample; and incubating the second matured APC peptide loaded sample with the second stimulated T cell sample, thereby obtaining a third stimulated T cell sample; and administering at least one T cell of the first stimulated T cell sample, the second stimulated T cell sample or the third stimulated T cell sample to a subject in need thereof.

In some embodiments, the method comprises sequencing cancer cell nucleic acids by whole genome sequencing or whole exome sequencing, thereby obtaining a first plurality of nucleic acid sequences comprising cancer cell nucleic acid sequences; and sequencing non-cancer cell nucleic acids by whole genome sequencing or whole exome sequencing, thereby obtaining a second plurality of nucleic acid sequences comprising non-cancer cell nucleic acid sequences. In some embodiments, the method comprises identifying a plurality of cancer specific nucleic acid sequences from a first plurality of nucleic acid sequences that are unique to cancer cells of the subject and that do not include nucleic acid sequences from a second plurality of nucleic acid sequences from non-cancer cells of the subject.

In some embodiments, the method further comprises selecting one or more subpopulation of cells from the expanded population of T cells prior to administering to the subject. In some embodiments, the selecting one or more subpopulation is performed by cell sorting based on expression of one or more cell surface markers provided herein. In some embodiments, the activated T cells may be sorted based on cell surface markers including but not limited to any one or more of the following: CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1, CD45RO, CCR7, FLT3LG, IL-6 and others.

In some embodiments, the method further comprises depleting one or more cells in the subject prior to administering the population of T cells.

In some embodiments, the one or more subpopulation of cells expressing a cell surface marker provided herein.

In some embodiments, the amino acid sequence of a peptide provided herein is validated by peptide sequencing. In some embodiments, the amino acid sequence a peptide provided herein is validated by mass spectrometry.

Also provided herein is a pharmaceutical composition comprising a T cell produced by expanding the T cell in the presence of an antigen presenting cell presenting one or more epitope sequence of any of Tables 1-8 and 11-14.

Also provided herein is library of polypeptides comprising epitope sequences or polynucleotides encoding the polypeptides, wherein each epitope sequence in the library is matched to a protein encoded by an HLA allele; and wherein each epitope sequence in the library is pre-validated to satisfy at least two or three or four of the following criteria: binds to a protein encoded by an HLA allele of a subject with cancer to be treated, is immunogenic according to an immunogenic assay, is presented by antigen presenting cells (APCs) according to a mass spectrometry assay, and stimulates T cells to be cytotoxic according to a cytotoxicity assay. In some embodiments, the library comprises one or two or more peptide sequences comprising an epitope sequence of any of Tables 1-8 and 11-14.

The peptides and polynucleotides provided herein can be for preparing antigen-specific T cells and include recombinant peptides and polynucleotides and synthetic peptides comprising epitopes, such as a tumor-specific neoepitopes, that have been identified and validated as binding to one or more MEC molecules, presented by the one or more MEC molecules, being immunogenic and/or capable of activating T cells to become cytotoxic. The peptides can be prepared for use in a method to prime T cells ex vivo. The peptides can be prepared for use in a method to activate T cells ex vivo. The peptides can be prepared for use in a method to expand antigen-specific T cells. The peptides can be prepared for use in a method to induce de novo CD8 T cell responses ex vivo. The peptides can be prepared for use in a method to induce de novo CD4 T cell responses ex vivo. The peptides can be prepared for use in a method to stimulate memory CD8 T cell responses ex vivo. The peptides can be prepared for use in a method to stimulate memory CD4 T cell responses ex vivo. The T cells can be obtained from a human subject. The T cells can be allogeneic T cells. The T cells can be T cell lines.

The epitopes can comprise at least 8 contiguous amino acids of an amino acid sequence encoded by the genome of a cancer cell. The epitopes can comprise from 8-12 contiguous amino acids of an amino acid sequence encoded by the genome of a cancer cell. The epitopes can comprise from 13-25 contiguous amino acids of an amino acid sequence encoded by the genome of a cancer cell. The epitopes can comprise from 8-50 contiguous amino acids of an amino acid sequence encoded by the genome of a cancer cell. In some embodiments, an epitope is from about 8 and about 30 amino acids in length. In some embodiments, an epitope is from about 8 to about 25 amino acids in length. In some embodiments, an epitope is from about 15 to about 24 amino acids in length. In some embodiments, an epitope is from about 9 to about 15 amino acids in length. In some embodiments, an epitope is 8 amino acids in length. In some embodiments, an epitope is 9 amino acids in length. In some embodiments, an epitope is 10 amino acids in length.

In some embodiments, a peptide containing an epitope is at most 500, at most 250, at most 150, at most 125, or at most 100 amino acids in length In some embodiments, a peptide containing an epitope is at least 8, at least 50, at least 100, at least 200, or at least 300 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 to about 500 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 to about 100 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 to about 50 amino acids in length. In some embodiments, a peptide containing an epitope is from about 15 to about 35 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 and about 15 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 and about 11 amino acids in length. In some embodiments, a peptide containing an epitope is 9 or 10 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 and about 30 amino acids in length. In some embodiments, a peptide containing an epitope is from about 8 to about 25 amino acids in length. In some embodiments, a peptide containing an epitope is from about 15 to about 24 amino acids in length. In some embodiments, a peptide containing an epitope is from about 9 to about 15 amino acids in length.

In some embodiments, a peptide containing an epitope has 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. In some embodiments, a peptide containing an epitope has 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. In some embodiments, a peptide containing an epitope comprises a first neoepitope peptide linked to at least a second neoepitope.

In some embodiments, a peptide contains a validated epitope from one or more of: ABL1, AC011997, ACVR2A, AFP, AKT1, ALK, ALPPL2, ANAPC1, APC, ARID1A, AR, AR-v7, ASCL2, β2M, BRAF, BTK, C15ORF40, CDH1, CLDN6, CNOT1, CT45A5, CTAG1B, DCT, DKK4, EEF1B2, EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM111B, FGFR3, FRG1B, GAGE1, GAGE10, GATA3, GBP3, HER2, IDH1, JAK1, KIT, KRAS, LMAN1, MABEB16, MAGEA1, MAGEA10, MAGEA4, MAGEA8, MAGEB17, MAGEB4, MAGEC1, MEK, MLANA, MLL2, MMP13, MSH3, MSH6, MYC, NDUFC2, NRAS, PAGE2, PAGES, PDGFRa, PIK3CA, PMEL, pol protein, POLE, PTEN, RAC1, RBM27, RNF43, RPL22, RUNX1, SEC31A, SEC63, SF3B1, SLC35F5, SLC45A2, SMAP1, SMAP1, SPOP, TFAM, TGFBR2, THAP5, TP53, TTK, TYR, UBR5, VHL, XPOT an EEF1DP3:FRY fusion polypeptide, an EGFR:SEPT14 fusion polypeptide, an EGFRVIII deletion polypeptide, an EML4:ALK fusion polypeptide, an NDRG1:ERG fusion polypeptide, an AC011997.1:LRRC69 fusion polypeptide, a RUNX1(ex5)-RUNX1T1fusion polypeptide, a TMPRSS2:ERG fusion polypeptide, a NAB:STAT6 fusion polypeptide, a NDRG1:ERG fusion polypeptide, a PML:RARA fusion polypeptide, a PPP1R1B:STARD3 fusion polypeptide, a MAD1L1:MAFK fusion polypeptide, a FGFR3:TAC fusion polypeptide, a FGFR3:TACC3 fusion polypeptide, a BCR:ABL fusion polypeptide, a C11orf95:RELA fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CD74:ROS1 fusion polypeptide, a CD74:ROS1 fusion polypeptide, ERVE-4: protease, ERVE-4: reverse transcriptase, ERVE-4: reverse transcriptase, ERVE-4: unknown, ERVH-2 matrix protein, ERVH-2: gag, ERVH-2: retroviral matrix, ERVH48-1: coat protein, ERVH48-1: syncytin, ERVI-1 envelope protein, ERVK-5 gag, ERVK-5 env, ERVK-5 pol, EBV A73, EBV BALF3, EBV BALF4, EBV BALF5, EBV BARF0, EBV LF2, EBV RPMS1, HPV-16, HPV-16 E7, and HPV-16 E6. In some embodiments, a neoepitope contains a mutation due to a mutational event in β2M, BTK, EGFR, GATA3, KRAS, MLL2, a TMPRSS2:ERG fusion polypeptide, or TP53 or Myc.

In some embodiments, an epitope binds a major histocompatibility complex (MEC) class I molecule. In some embodiments, an epitope binds an MEC class I molecule with a binding affinity of about 500 nM or less. In some embodiments an epitope binds an MEC class I molecule with a binding affinity of about 250 nM or less. In some embodiments, an epitope binds an MEC class I molecule with a binding affinity of about 150 nM or less. In some embodiments, an epitope binds an MEC class I molecule with a binding affinity of about 50 nM or less.

In some embodiments, an epitope binds an binds MEC class I molecule and a peptide containing the class I epitope binds to an MEC class II molecule.

In some embodiments, an epitope binds an MEC class II molecule. In some embodiments, an epitope binds to human leukocyte antigen (HLA)-A, -B, -C, -DP, -DQ, or -DR. In some embodiments, an epitope binds an MEC class II molecule with a binding affinity of 1000 nM or less. In some embodiments, an epitope binds MEC class II with a binding affinity of 500 nM or less. In some embodiments an epitope binds an MEC class II molecule with a binding affinity of about 250 nM or less. In some embodiments, an epitope binds an MEC class II molecule with a binding affinity of about 150 nM or less. In some embodiments, an epitope binds an MEC class II molecule with a binding affinity of about 50 nM or less.

In some embodiments, a peptide containing a validated epitope further comprises one or more amino acids flanking the C-terminus of the epitope. In some embodiments, a peptide containing a validated epitope further comprises one or more amino acids flanking the N-terminus of the epitope. In some embodiments, a peptide containing a validated epitope further comprises one or more amino acids flanking the C-terminus of the epitope and one or more amino acids flanking the N-terminus of the epitope. In some embodiments, the flanking amino acids are not native flanking amino acids. In some embodiments, a first epitope used in a method described herein binds an MEC class I molecule and a second epitope binds an MHC class II molecule. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases in vivo half-life of the peptide. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases cellular targeting by the peptide. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases cellular uptake of the peptide. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases peptide processing. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases MHC affinity of the epitope. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases MEC stability of the epitope. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases presentation of the epitope by an MHC class I molecule, and/or an MHC class II molecule.

In some embodiments, sequencing methods are used to identify tumor specific mutations. Any suitable sequencing method can be used according to the invention, for example, Next Generation Sequencing (NGS) technologies. Third Generation Sequencing methods might substitute for the NGS technology in the future to speed up the sequencing step of the method. For clarification purposes: the terms “Next Generation Sequencing” or “NGS” in the context of the present invention mean all novel high throughput sequencing technologies which, in contrast to the “conventional” sequencing methodology known as Sanger chemistry, read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces. Such NGS technologies (also known as massively parallel sequencing technologies) are able to deliver nucleic acid sequence information of a whole genome, exome, transcriptome (all transcribed sequences of a genome) or methylome (all methylated sequences of a genome) in very short time periods, e.g. within 1-2 weeks, for example, within 1-7 days or within less than 24 hours and allow, in principle, single cell sequencing approaches. Multiple NGS platforms which are commercially available or which are mentioned in the literature can be used in the context of the invention e.g. those described in detail in WO 2012/159643.

In some embodiments, a peptide containing a validated epitope is linked to the at least second peptide, such as by a poly-glycine or poly-serine linker. In some embodiments, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids. In some embodiments, a peptide containing a validated epitope further comprises a modification which increases cellular targeting to specific organs, tissues, or cell types. In some embodiments, a peptide containing a validated epitope comprises an antigen presenting cell targeting moiety or marker. In some embodiments, the antigen presenting cells are dendritic cells. In some embodiments, the dendritic cells are targeted using DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, Clec9a, or CD1a marker. In some embodiments, the dendritic cells are targeted using the CD141, DEC205, Clec9a, or XCR1 marker. In some embodiments, the dendritic cells are autologous cells. In some embodiments, one or more of the dendritic cells are bound to a T cell.

In some embodiments, the method described herein comprises large scale manufacture of and storage of HLA-matched peptides corresponding to shared antigens for treatment of a cancer or a tumor.

In some embodiments, the method described herein comprises treatment methods, comprising administering to a subject with cancer antigen-specific T cell that are specific to a validated epitope selected from the HLA matched peptide repertoire presented in any of Tables 1-8 and 11-14. In some embodiments, epitope-specific T cells are administered to the patient by infusion. In some embodiments, the T cells are administered to the patient by direct intravenous injection. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell.

The methods of the disclosure can be used to treat any type of cancer known in the art. In some embodiments, a method of treating cancer comprises treating breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer, metastatic melanoma, thymoma, lymphoma, sarcoma, mesothelioma, renal cell carcinoma, stomach cancer, gastric cancer, ovarian cancer, NHL, leukemia, uterine cancer, colon cancer, bladder cancer, kidney cancer or endometrial cancer. In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, head and neck cancer, colorectal cancer, rectal cancer, soft-tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, Hairy cell leukemia, chronic myeloblasts leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, Meigs' syndrome. Non-limiting examples of cancers to be treated by the methods of the present disclosure can include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, a cancer to be treated by the methods of the present disclosure include, for example, carcinoma, squamous carcinoma (for example, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary). In some embodiments, a cancer to be treated by the methods of the present disclosure further include sarcomata (for example, myogenic sarcoma), leukosis, neuroma, melanoma, and lymphoma. In some embodiments, a cancer to be treated by the methods of the present disclosure is breast cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is triple negative breast cancer (TNBC). In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is prostate cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is colorectal cancer. In some embodiments, a patient or population of patients to be treated with a pharmaceutical composition of the present disclosure have a solid tumor. In some embodiments, a solid tumor is a melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma. In some embodiments, a patient or population of patients to be treated with a pharmaceutical composition of the present disclosure have a hematological cancer. In some embodiments, the patient has a hematological cancer such as diffuse large B cell lymphoma (“DLBCL”), Hodgkin's lymphoma (“HL”), Non-Hodgkin's lymphoma (“NHL”), Follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), or Multiple myeloma (“MM”). In some embodiments, a patient or population of patients to be treated having the cancer selected from the group consisting of ovarian cancer, lung cancer and melanoma.

The pharmaceutical compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). In some embodiments, at least one or more chemotherapeutic agents may be administered in addition to the pharmaceutical composition comprising an immunogenic therapy. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents. In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the pharmaceutical compositions can be administered to a subject having a disease or condition. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

In some embodiments, the methods for treatment include one or more rounds of leukapheresis prior to transplantation of T cells. The leukapheresis may include collection of peripheral blood mononuclear cells (PBMCs). Leukapheresis may include mobilizing the PBMCs prior to collection. Alternatively, non-mobilized PBMCs may be collected. A large volume of PBMCs may be collected from the subject in one round. Alternatively, the subject may undergo two or more rounds of leukapheresis. The volume of apheresis may be dependent on the number of cells required for transplant. For instance, 12-15 liters of non-mobilized PBMCs may be collected from a subject in one round. The number of PBMCs to be collected from a subject may be between 1×10⁸to 5×10¹⁰cells. The number of PBMCs to be collected from a subject may be 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰or 5×10¹⁰cells. The minimum number of PBMCs to be collected from a subject may be 1×10⁶/kg of the subject's weight. The minimum number of PBMCs to be collected from a subject may be 1×10⁶/kg, 5×10⁶/kg, 1×10⁷/kg, 5×10⁷/kg, 1×10⁸/kg, 5×10⁸/kg of the subject's weight.

A single infusion may comprise a dose between 1×10⁶cells per square meter body surface of the subject (cells/m²) and 5×10⁹cells/m². A single infusion may comprise between about 2.5×10⁶to about 5×10⁹cells/m². A single infusion may comprise between at least about 2.5×10⁶cells/m². A single infusion may comprise between at most 5×10⁹cells/m². A single infusion may comprise between 1×10⁶to 2.5×10⁶, 1×10⁶to 5×10⁶, 1×10⁶to 7.5×10⁶, 1×10⁶to 1×10⁷, 1×10⁶to 5×10⁷, 1×10⁶to 7.5×10⁷, 1×10⁶to 1×10⁸, 1×10⁶to 2.5×10⁸, 1×10⁶to 5×10⁸, 1×10⁶to 1×10⁹, 1×10⁶to 5×10⁹, 2.5×10⁶to 5×10⁶, 2.5×10⁶to 7.5×10⁶, 2.5×10⁶to 1×10⁷, 2.5×10⁶to 5×10⁷, 2.5×10⁶to 7.5×10⁷, 2.5×10⁶to 1×10⁸, 2.5×10⁶to 2.5×10⁸, 2.5×10⁶to 5×10⁸, 2.5×10⁶to 1×10⁹, 2.5×10⁶to 5×10⁹, 5×10⁶to 7.5×10⁶, 5×10⁶to 1×10⁷, 5×10⁶to 5×10⁷, 5×10⁶to 7.5×10⁷, 5×10⁶to 1×10⁸, 5×10⁶to 2.5×10⁸, 5×10⁶to 5×10⁸, 5×10⁶to 1×10⁹, 5×10⁶to 5×10⁹, 7.5×10⁶to 1×10⁷, 7.5×10⁶to 5×10⁷, 7.5×10⁶to 7.5×10⁷, 7.5×10⁶to 1×10⁸, 7.5×10⁶to 2.5×10⁸, 7.5×10⁶to 5×10⁸, 7.5×10⁶to 1×10⁹, 7.5×10⁶to 5×10⁹, 1×10⁷to 5×10⁷, 1×10⁷to 7.5×10⁷, 1×10⁷to 1×10⁸, 1×10⁷to 2.5×10⁸, 1×10⁷to 5×10⁸, 1×10⁷to 1×10⁹, 1×10⁷to 5×10⁹, 5×10⁷to 7.5×10⁷, 5×10⁷to 1×10⁸, 5×10⁷to 2.5×10⁸, 5×10⁷to 5×10⁸, 5×10⁷to 1×10⁹, 5×10⁷to 5×10⁹, 7.5×10⁷to 1×10⁸, 7.5×10⁷to 2.5×10⁸, 7.5×10⁷to 5×10⁸, 7.5×10⁷to 1×10⁹, 7.5×10⁷to 5×10⁹, 1×10⁸to 2.5×10⁸, 1×10⁸to 5×10⁸, 1×10⁸to 1×10⁹, 1×10⁸to 5×10⁹, 2.5×10⁸to 5×10⁸, 2.5×10⁸to 1×10⁹, 2.5×10⁸to 5×10⁹, 5×10⁸to 1×10⁹, 5×10⁸to 5×10⁹, or 1×10⁹to 5×10⁹cells/m². A single infusion may comprise between 1×10⁶cells/m², 2.5×10⁶cells/m², 5×10⁶cells/m², 7.5×10⁶cells/m², 1×10⁷cells/m², 5×10⁷cells/m², 7.5×10⁷cells/m², 1×10⁸cells/m², 2.5×10⁸cells/m², 5×10⁸cells/m², 1×10⁹cells/m², or 5×10⁹cells/m².

The methods may include administering chemotherapy to a subject including lymphodepleting chemotherapy using high doses of myeloablative agents. In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the first or subsequent dose. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, 7, 8, 9 or 10 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 10 days prior, such as no more than 9, 8, 7, 6, 5, 4, 3, or 2 days prior, to the first or subsequent dose.

In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered between 0.3 grams per square meter of the body surface of the subject (g/m²) and 5 g/m²cyclophosphamide. In some cases, the amount of cyclophosphamide administered to a subject is about at least 0.3 g/m². In some cases, the amount of cyclophosphamide administered to a subject is about at most 5 g/m². In some cases, the amount of cyclophosphamide administered to a subject is about 0.3 g/m²to 0.4 g/m², 0.3 g/m²to 0.5 g/m², 0.3 g/m²to 0.6 g/m², 0.3 g/m²to 0.7 g/m², 0.3 g/m²to 0.8 g/m², 0.3 g/m²to 0.9 g/m², 0.3 g/m²to 1 g/m², 0.3 g/m²to 2 g/m², 0.3 g/m²to 3 g/m², 0.3 g/m²to 4 g/m², 0.3 g/m²to 5 g/m², 0.4 g/m²to 0.5 g/m², 0.4 g/m²to 0.6 g/m², 0.4 g/m²to 0.7 g/m², 0.4 g/m²to 0.8 g/m², 0.4 g/m²to 0.9 g/m², 0.4 g/m²to 1 g/m², 0.4 g/m²to 2 g/m², 0.4 g/m²to 3 g/m², 0.4 g/m²to 4 g/m², 0.4 g/m²to 5 g/m², 0.5 g/m²to 0.6 g/m², 0.5 g/m²to 0.7 g/m², 0.5 g/m²to 0.8 g/m², 0.5 g/m²to 0.9 g/m², 0.5 g/m²to 1 g/m², 0.5 g/m²to 2 g/m², 0.5 g/m²to 3 g/m², 0.5 g/m²to 4 g/m², 0.5 g/m²to 5 g/m², 0.6 g/m²to 0.7 g/m², 0.6 g/m²to 0.8 g/m², 0.6 g/m²to 0.9 g/m², 0.6 g/m²to 1 g/m², 0.6 g/m²to 2 g/m², 0.6 g/m²to 3 g/m², 0.6 g/m²to 4 g/m², 0.6 g/m²to 5 g/m², 0.7 g/m²to 0.8 g/m², 0.7 g/m²to 0.9 g/m², 0.7 g/m²to 1 g/m², 0.7 g/m²to 2 g/m², 0.7 g/m²to 3 g/m², 0.7 g/m²to 4 g/m², 0.7 g/m²to 5 g/m², 0.8 g/m²to 0.9 g/m², 0.8 g/m²to 1 g/m², 0.8 g/m²to 2 g/m², 0.8 g/m²to 3 g/m², 0.8 g/m²to 4 g/m², 0.8 g/m²to 5 g/m², 0.9 g/m²to 1 g/m², 0.9 g/m²to 2 g/m², 0.9 g/m²to 3 g/m², 0.9 g/m²to 4 g/m², 0.9 g/m²to 5 g/m², 1 g/m²to 2 g/m², 1 g/m²to 3 g/m², 1 g/m²to 4 g/m², 1 g/m²to 5 g/m², 2 g/m²to 3 g/m², 2 g/m²to 4 g/m², 2 g/m²to 5 g/m², 3 g/m²to 4 g/m², 3 g/m²to 5 g/m², or 4 g/m²to 5 g/m². In some cases, the amount of cyclophosphamide administered to a subject is about 0.3 g/m², 0.4 g/m², 0.5 g/m², 0.6 g/m², 0.7 g/m², 0.8 g/m², 0.9 g/m², 1 g/m², 2 g/m², 3 g/m², 4 g/m², or 5 g/m². In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide.

In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 milligrams per square meter of the body surface of the subject (mg/m²) and 100 mg/m². In some cases, the amount of fludarabine administered to a subject is about at least 1 mg/m². In some cases, the amount of fludarabine administered to a subject is about at most 100 mg/m². In some cases, the amount of fludarabine administered to a subject is about 1 mg/m²to 5 mg/m², 1 mg/m²to 10 mg/m², 1 mg/m²to 15 mg/m², 1 mg/m²to 20 mg/m², 1 mg/m²to 30 mg/m², 1 mg/m²to 40 mg/m², 1 mg/m²to 50 mg/m², 1 mg/m²to 70 mg/m², 1 mg/m²to 90 mg/m², 1 mg/m²to 100 mg/m², 5 mg/m²to 10 mg/m², 5 mg/m²to 15 mg/m², 5 mg/m²to 20 mg/m², 5 mg/m²to 30 mg/m², 5 mg/m²to 40 mg/m², 5 mg/m²to 50 mg/m², 5 mg/m²to 70 mg/m², 5 mg/m²to 90 mg/m², 5 mg/m²to 100 mg/m², 10 mg/m²to 15 mg/m², 10 mg/m²to 20 mg/m², 10 mg/m²to 30 mg/m², 10 mg/m²to 40 mg/m², 10 mg/m²to 50 mg/m², 10 mg/m²to 70 mg/m², 10 mg/m²to 90 mg/m², 10 mg/m²to 100 mg/m², 15 mg/m²to 20 mg/m², 15 mg/m²to 30 mg/m², 15 mg/m²to 40 mg/m², 15 mg/m²to 50 mg/m², 15 mg/m²to 70 mg/m², 15 mg/m²to 90 mg/m², 15 mg/m²to 100 mg/m², 20 mg/m²to 30 mg/m², 20 mg/m²to 40 mg/m², 20 mg/m²to 50 mg/m², 20 mg/m²to 70 mg/m², 20 mg/m²to 90 mg/m², 20 mg/m²to 100 mg/m², 30 mg/m²to 40 mg/m², 30 mg/m²to 50 mg/m², 30 mg/m²to 70 mg/m², 30 mg/m²to 90 mg/m², 30 mg/m²to 100 mg/m², 40 mg/m²to 50 mg/m², 40 mg/m²to 70 mg/m², 40 mg/m²to 90 mg/m², 40 mg/m²to 100 mg/m², 50 mg/m²to 70 mg/m², 50 mg/m²to 90 mg/m², 50 mg/m²to 100 mg/m², 70 mg/m²to 90 mg/m², 70 mg/m²to 100 mg/m², or 90 mg/m²to 100 mg/m². In some cases, the amount of fludarabine administered to a subject is about 1 mg/m², 5 mg/m², 10 mg/m², 15 mg/m², 20 mg/m², 30 mg/m², 40 mg/m², 50 mg/m², 70 mg/m², 90 mg/m², or 100 mg/m². In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. For example, in some instances, the agent, e.g., fludarabine, is administered between or between about 1 and 5 times, such as between or between about 3 and 5 times. In some embodiments, such plurality of doses is administered in the same day, such as 1 to 5 times or 3 to 5 times daily.

In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 400 mg/m²of cyclophosphamide and one or more doses of 20 mg/m²fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 500 mg/m²of cyclophosphamide and one or more doses of 25 mg/m²fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 600 mg/m²of cyclophosphamide and one or more doses of 30 mg/m²fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 700 mg/m²of cyclophosphamide and one or more doses of 35 mg/m²fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 700 mg/m²of cyclophosphamide and one or more doses of 40 mg/m²fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 800 mg/m²of cyclophosphamide and one or more doses of 45 mg/m²fludarabine prior to the first or subsequent dose of T cells.

Fludarabine and cyclophosphamide may be administered on alternative days. In some cases, fludarabine and cyclophosphamide may be administered concurrently. In some cases, an initial dose of fludarabine is followed by a dose of cyclophosphamide. In some cases, an initial dose of cyclophosphamide may be followed by an initial dose of fludarabine. In some examples, a treatment regimen may include treatment of a subject with an initial dose of fludarabine 10 days prior to the transplant, followed by treatment with an initial dose of cyclophosphamide administered 9 days prior to the cell transplant, concurrently with a second dose of fludarabine. In some examples, a treatment regimen may include treatment of a subject with an initial dose of fludarabine 8 days prior to the transplant, followed by treatment with an initial dose of cyclophosphamide administered 7 days prior to the transplant concurrently with a second dose of fludarabine.

In some embodiments, a peptide comprises an epitope sequence according to any one of Tables 1-8 and 11-14. In some embodiments, a peptide comprises an epitope sequence according to Table 1. In some embodiments, a peptide comprises an epitope sequence according to Table 2. In some embodiments, a peptide comprises an epitope sequence according to Table 3. In some embodiments, a peptide comprises an epitope sequence according to Table 4A. In some embodiments, a peptide comprises an epitope sequence according to Table 4B. In some embodiments, a peptide comprises an epitope sequence according to Table 4C. In some embodiments, a peptide comprises an epitope sequence according to Table 4D. In some embodiments, a peptide comprises an epitope sequence according to Table 4E. In some embodiments, a peptide comprises an epitope sequence according to Table 4F. In some embodiments, a peptide comprises an epitope sequence according to Table 4G. In some embodiments, a peptide comprises an epitope sequence according to Table 4H. In some embodiments, a peptide comprises an epitope sequence according to Table 41. In some embodiments, a peptide comprises an epitope sequence according to Table 4J. In some embodiments, a peptide comprises an epitope sequence according to Table 4K. In some embodiments, a peptide comprises an epitope sequence according to Table 4L. In some embodiments, a peptide comprises an epitope sequence according to Table 4M. In some embodiments, a peptide comprises an epitope sequence according to Table 5. In some embodiments, a peptide comprises an epitope sequence according to Table 6. In some embodiments, a peptide comprises an epitope sequence according to Table 7. In some embodiments, a peptide comprises an epitope sequence according to Table 8. In some embodiments, a peptide comprises an epitope sequence according to Table 11. In some embodiments, a peptide comprises an epitope sequence according to Table 12. In some embodiments, a peptide comprises an epitope sequence according to Table 13. In some embodiments, a peptide comprises an epitope sequence according to Table 14.

TABLE 1

TABLE 1A POINT MUTATION

Amino Acid

Peptides (Binding HLA allele
Exemplary

Gene
Alteration
Mutation Sequence Context
example(s))
Diseases

KRAS
G12C
MTEYKLVVVGACGVGKSA
KLVVVGACGV (SEQ ID
BRCA, CESC,

LTIQLIQNHFVDEYDPTIEDS
NO: 154)(A02.01)
CRC, HNSC,

YRKQVVIDGETCLLDILDT
LVVVGACGV (SEQ ID
LUAD, PAAD,

AGQE (SEQ ID NO: 8)
NO: 155)(A02.01)
UCEC

VVGACGVGK (SEQ ID

NO: 156)(A03.01, A11.01)

VVVGACGVGK (SEQ ID

NO: 157)(A03.01)

KRAS
G12D
MTEYKLVVVGADGVGKSA
VVGADGVGK (SEQ ID
BLCA, BRCA,

LTIQLIQNHFVDEYDPTIEDS
NO: 158)(A11.01)
CESC, CRC,

YRKQVVIDGETCLLDILDT
VVVGADGVGK (SEQ ID
GBM, HNSC,

AGQE (SEQ ID NO: 9)
NO: 159)(A11.01)
KIRP, LIHC,

KLVVVGADGV (SEQ ID
LUAD, PAAD,

NO: 160)(A02.01)
SKCM, UCEC

LVVVGADGV (SEQ ID

NO: 161)(A02.01)

KRAS
G12V
MTEYKLVVVGAVGVGKSA
KLVVVGAVGV (SEQ ID
BRCA, CESC,

LTIQLIQNHFVDEYDPTIEDS
NO: 162)(A02.01)
CRC, LUAD,

YRKQVVIDGETCLLDILDT
LVVVGAVGV (SEQ ID
PAAD, THCA,

AGQE (SEQ ID NO: 10)
NO: 163)(A02.01)
UCEC

VVGAVGVGK (SEQ ID

NO: 164)(A03.01, A11.01)

VVVGAVGVGK (SEQ ID

NO: 5)(A03.01, A11.01)

KRAS
Q61H
AGGVGKSALTIQLIQNHFV
ILDTAGHEEY (SEQ ID
CRC, LUSC,

DEYDPTIEDSYRKQVVIDGE
NO: 165)(A01.01)
PAAD, SKCM,

TCLLDILDTAGHEEYSAMR

UCEC

DQYMRTGEGFLCVFAINNT

KSFEDIHHYREQIKRVKDSE

DVPM (SEQ ID NO: 11)

KRAS
Q61L
AGGVGKSALTIQLIQNHFV
ILDTAGLEEY (SEQ ID
CRC, GBM,

DEYDPTIEDSYRKQVVIDGE
NO: 166)(A01.01)
HNSC, LUAD,

TCLLDILDTAGLEEYSAMR
LLDILDTAGL (SEQ ID
SKCM, UCEC

DQYMRTGEGFLCVFAINNT
NO: 167)(A02.01)

KSFEDIHHYREQIKRVKDSE

DVPM (SEQ ID NO: 12)

NRAS
Q61K
AGGVGKSALTIQLIQNHFV
ILDTAGKEEY (SEQ ID
BLCA, CRC,

DEYDPTIEDSYRKQVVIDGE
NO: 168)(A01.01)
LIHC, LUAD,

TCLLDILDTAGKEEYSAMR

LUSC, SKCM,

DQYMRTGEGFLCVFAINNS

THCA, UCEC

KSFADINLYREQIKRVKDSD

DVPM (SEQ ID NO: 13)

NRAS
Q61R
AGGVGKSALTIQLIQNHFV
ILDTAGREEY (SEQ ID
BLCA, CRC,

DEYDPTIEDSYRKQVVIDGE
NO: 169)(A01.01)
LUSC, PAAD,

TCLLDILDTAGREEYSAMR

PRAD, SKCM,

DQYMRTGEGFLCVFAINNS

THCA, UCEC

KSFADINLYREQIKRVKDSD

DVPM (SEQ ID NO: 14)

BTK
C481S
MIKEGSMSEDEFIEEAKVM
EYMANGSLL (SEQ ID NO:
CLL

MNLSHEKLVQLYGVCTKQ
170)(A24.02)

RPIFIITEYMANGSLLNYLR
MANGSLLNY (SEQ ID

EMRHRFQTQQLLEMCKDV
NO: 171)(A01.01, A03.01,

CEAMEYLESKQFLHRDLA
A11.01)

ARNCLVND (SEQ ID NO:
MANGSLLNYL (SEQ ID

15)
NO: 172)(A02.01, B07.02,

B08.01)

SLLNYLREM (SEQ ID NO:

173)(A02.01, B07.02,

B08.01)

YMANGSLLN (SEQ ID

NO: 174)(A02.01)

YMANGSLLNY (SEQ ID

NO: 175)(A01.01, A03.01,

A11.01)

EGFR
S492R
SLNITSLGLRSLKEISDGDVI
IIRNRGENSCK (SEQ ID
CRC

ISGNKNLCYANTINWKKLF
NO: 176)(A03.01)

GTSGQKTKIIRNRGENSCK

ATGQVCHALCSPEGCWGP

EPRDCVSCRNVSRGRECVD

KCNLL (SEQ ID NO: 16)

EGFR
T790M
IPVAIKELREATSPKANKEI
CLTSTVQLIM (SEQ ID
NSCLC, PRAD

LDEAYVMASVDNPHVCRL
NO: 177)(A01.01, A02.01)

LGICLTSTVQLIMQLMPFGC
IMQLMPFGC (SEQ ID NO:

LLDYVREHKDNIGSQYLLN
178)(A02.01)

WCVQIAKGMNYLEDRRLV
IMQLMPFGCL (SEQ ID

HRDLAA (SEQ ID NO: 17)
NO: 179)(A02.01, A24.02,

B08.01)

LIMQLMPFG (SEQ ID NO:

180)(A02.01)

LIMQLMPFGC (SEQ ID

NO: 181)(A02.01)

LTSTVQLIM (SEQ ID NO:

182)(A01.01)

MQLMPFGCL (SEQ ID NO:

183)(A02.01, B07.02,

B08.01)

MQLMPFGCLL (SEQ ID

NO: 184)(A02.01, A24.02,

B08.01)

QLIMQLMPF (SEQ ID NO:

185)(A02.01, A24.02,

B08.01)

QLIMQLMPFG (SEQ ID

NO: 186)(A02.01)

STVQLIMQL (SEQ ID NO:

187)(A02.01)

VQLIMQLMPF (SEQ ID

NO: 188)(A02.01, A24.02,

B08.01)

ABL1
E255K
VADGLITTLHYPAPKRNKP
GQYGKVYEG (SEQ ID
Chronic

TVYGVSPNYDKWEMERTD
NO: 189)(A02.01)
myeloid

ITMKHKLGGGQYGKVYEG
GQYGKVYEGV (SEQ ID
leukemia

VWKKYSLTVAVKTLKEDT
NO: 190)(A02.01)
(CML), Acute

MEVEEFLKEAAVMKEIKHP
KLGGGQYGK (SEQ ID
lymphocytic

NLVQLLGVC (SEQ ID NO:
NO: 191)(A03.01)
leukemia

18)
KLGGGQYGKV (SEQ ID
(ALL),

NO: 192)(A02.01)
Gastrointestinal

KVYEGVWKK (SEQ ID
stromal tumors

NO: 193)(A02.01, A03.01)
(GIST)

KVYEGVWKKY (SEQ ID

NO: 194)(A03.01)

QYGKVYEGV (SEQ ID

NO: 195)(A24.02)

QYGKVYEGVW (SEQ ID

NO: 196)(A24.02)

ABL1
E255V
VADGLITTLHYPAPKRNKP
GQYGVVYEG (SEQ ID
Chronic

TVYGVSPNYDKWEMERTD
NO: 197)(A02.01)
myeloid

ITMKHKLGGGQYGVVYEG
GQYGVVYEGV (SEQ ID
leukemia

VWKKYSLTVAVKTLKEDT
NO: 198)(A02.01)
(CML), Acute

MEVEEFLKEAAVMKEIKHP
KLGGGQYGV (SEQ ID
lymphocytic

NLVQLLGVC (SEQ ID NO:
NO: 199)(A02.01)
leukemia

19)
KLGGGQYGVV (SEQ ID
(ALL),

NO: 200)(A02.01)
Gastrointestinal

QYGVVYEGV (SEQ ID
stromal tumors

NO: 201)(A24.02)
(GIST)

QYGVVYEGVW (SEQ ID

NO: 202)(A24.02)

VVYEGVWKK (SEQ ID

NO: 203)(A02.01, A03.01)

VVYEGVWKKY (SEQ ID

NO: 204)(A03.01)

ABL1
M351T
LLGVCTREPPFYIITEFMTY
ATQISSATEY (SEQ ID NO:
Chronic

GNLLDYLRECNRQEVNAV
205)(A01.01)
myeloid

VLLYMATQISSATEYLEKK
ISSATEYLEK (SEQ ID NO:
leukemia

NFIHRDLAARNCLVGENHL
206)(A03.01)
(CML), Acute

VKVADFGLSRLMTGDTYT
SSATEYLEK (SEQ ID NO:
lymphocytic

AHAGAKF (SEQ ID NO: 20)
207)(A03.01)
leukemia

TQISSATEYL (SEQ ID NO:
(ALL),

208)(A02.01)
Gastrointestinal

YMATQISSAT (SEQ ID
stromal tumors

NO: 209)(A02.01)
(GIST)

ABL1
T315I
SLTVAVKTLKEDTMEVEEF
FYIIIEFMTY (SEQ ID NO:
Chronic

LKEAAVMKEIKHPNLVQLL
210)(A24.02)
myeloid

GVCTREPPFYIIIEFMTYGN
IIEFMTYGNL (SEQ ID NO:
leukemia

LLDYLRECNRQEVNAVVL
211)(A02.01)
(CML), Acute

LYMATQISSAMEYLEKKNF
IIIEFMTYG (SEQ ID NO:
lymphocytic

IHRDLA (SEQ ID NO: 21)
212)(A02.01)
leukemia

IIIEFMTYGN (SEQ ID NO:
(ALL),

213)(A02.01)
Gastrointestinal

YIIIEFMTYG (SEQ ID NO:
stromal tumors

214)(A02.01)
(GIST)

ABL1
Y253H
STVADGLITTLHYPAPKRN
GQHGEVYEGV (SEQ ID
Chronic

KPTVYGVSPNYDKWEMER
NO: 215)(A02.01)
myeloid

TDITMKHKLGGGQHGEVY
KLGGGQHGEV (SEQ ID
leukemia

EGVWKKYSLTVAVKTLKE
NO: 216)(A02.01)
(CML), Acute

DTMEVEEFLKEAAVMKEIK

lymphocytic

HPNLVQLLG (SEQ ID NO:

leukemia

22)

(ALL),

Gastrointestinal

stromal tumors

(GIST)

ALK
G1269A
SSLAMLDLLHVARDIACGC
KIADFGMAR (SEQ ID NO:
NSCLC

QYLEENHFIHRDIAARNCL
217)(A03.01)

LTCPGPGRVAKIADFGMAR
RVAKIADFGM (SEQ ID

DIYRASYYRKGGCAMLPV
NO: 218)(A02.01, B07.02)

KWMPPEAFMEGIFTSKTDT

WSFGVLL (SEQ ID NO: 23)

ALK
L1196M
QVAVKTLPEVCSEQDELDF
FILMELMAGG (SEQ ID
NSCLC

LMEALIISKFNHQNIVRCIG
NO: 219)(A02.01)

VSLQSLPRFILMELMAGGD
ILMELMAGG (SEQ ID NO:

LKSFLRETRPRPSQPSSLAM
220)(A02.01)

LDLLHVARDIACGCQYLEE
ILMELMAGGD (SEQ ID

NHFI (SEQ ID NO: 24)
NO: 221)(A02.01)

LMELMAGGDL (SEQ ID

NO: 222)(A02.01)

LPRFILMEL (SEQ ID NO:

223)(B07.02, B08.01)

LPRFILMELM (SEQ ID

NO: 224)(B07.02)

LQSLPRFILM (SEQ ID NO:

225)(A02.01, B08.01)

SLPRFILMEL (SEQ ID NO:

226)(A02.01, A24.02,

B07.02, B08.01)

BRAF
V600E
MIKLIDIARQTAQGMDYLH
LATEKSRWS (SEQ ID NO:
CRC, GBM,

AKSIIHRDLKSNNIFLHEDL
227)(A02.01, B08.01)
KIRP, LUAD,

TVKIGDFGLATEKSRWSGS
LATEKSRWSG (SEQ ID
SKCM, THCA

HQFEQLSGSILWMAPEVIR
NO: 228)(A02.01, B08.01)

MQDKNPYSFQSDVYAFGIV

LYELM (SEQ ID NO: 25)

EEF1B2
S43G
MGFGDLKSPAGLQVLNDY
GPPPADLCHAL (SEQ ID
BLCA, KIRP,

LADKSYIEGYVPSQADVAV
NO: 229)(B07.02)
PRAD, SKCM

FEAVSGPPPADLCHALRWY

NHIKSYEKEKASLPGVKKA

LGKYGPADVEDTTGSGAT

(SEQ ID NO: 26)

ERBB3
V104M
ERCEVVMGNLEIVLTGHNA

CRC, Stomach

DLSFLQWIREVTGYVLVA

Cancer

MNEFSTLPLPNLRMVRGTQ

VYDGKFAIFVMLNYNTNSS

HALRQLRLTQLTEILSGGV

YIEKNDK (SEQ ID NO: 27)

ESR1
D538G
HLMAKAGLTLQQQHQRLA
GLLLEMLDA (SEQ ID NO:
Breast Cancer

QLLLILSHIRHMSNKGMEH
230)(A02.01)

LYSMKCKNVVPLYGLLLE
LYGLLLEML (SEQ ID NO:

MLDAHRLHAPTSRGGASV
231)(A24.02)

EETDQSHLATAGSTSSHSL
NVVPLYGLL (SEQ ID NO:

QKYYITGEA (SEQ ID NO:
232)(A02.01)

28)
PLYGLLLEM (SEQ ID NO:

233)(A02.01)

PLYGLLLEML (SEQ ID

NO: 234)(A02.01, A24.02)

VPLYGLLLEM (SEQ ID

NO: 235)(B07.02)

VVPLYGLLL (SEQ ID NO:

236)(A02.01, A24.02)

ESR1
S463P
NQGKCVEGMVEIFDMLLA
FLPSTLKSL (SEQ ID NO:
Breast Cancer

TSSRFRMMNLQGEEFVCLK
237)(A02.01, A24.02,

SIILLNSGVYTFLPSTLKSLE
B08.01)

EKDHIHRVLDKITDTLIHLM
GVYTFLPST (SEQ ID NO:

AKAGLTLQQQHQRLAQLL
238)(A02.01)

LILSH (SEQ ID NO: 29)
GVYTFLPSTL (SEQ ID

NO. 239)(A02.01, A24.02)

TFLPSTLKSL (SEQ ID NO:

240)(A24.02)

VYTFLPSTL (SEQ ID NO:

241)(A24.02)

YTFLPSTLK (SEQ ID NO:

242)(A03.01)

ESR1
Y537C
IHLMAKAGLTLQQQHQRL
NVVPLCDLL (SEQ ID NO:
Breast Cancer

AQLLLILSHIRHMSNKGME
243)(A02.01)

HLYSMKCKNVVPLCDLLL
NVVPLCDLLL (SEQ ID

EMLDAHRLHAPTSRGGAS
NO: 244)(A02.01)

VEETDQSHLATAGSTSSHS
PLCDLLLEM (SEQ ID NO:

LQKYYITGE (SEQ ID NO:
245)(A02. 01)

30)
PLCDLLLEML (SEQ ID

NO: 246)(A02.01)

VPLCDLLLEM (SEQ ID

NO: 247)(B07.02)

VVPLCDLLL (SEQ ID NO:

248)(A02.01, A24.02)

ESR1
Y537N
IHLMAKAGLTLQQQHQRL
NVVPLNDLL (SEQ ID NO:
Breast Cancer

AQLLLILSHIRHMSNKGME
249)(A02.01)

HLYSMKCKNVVPLNDLLL
NVVPLNDLLL (SEQ ID

EMLDAHRLHAPTSRGGAS
NO: 250)(A02.01)

VEETDQSHLATAGSTSSHS
PLNDLLLEM (SEQ ID NO:

LQKYYITGE (SEQ ID NO:
251)(A02.01)

31)
PLNDLLLEML (SEQ ID

NO: 252)(A02.01)

VPLNDLLLEM (SEQ ID

NO: 253)(B07.02)

ESR1
Y537S
IHLMAKAGLTLQQQHQRL
NVVPLSDLL (SEQ ID NO:
Breast Cancer

AQLLLILSHIRHMSNKGME
254)(A02.01)

HLYSMKCKNVVPLSDLLLE
NVVPLSDLLL (SEQ ID

MLDAHRLHAPTSRGGASV
NO: 255)(A02.01)

EETDQSHLATAGSTSSHSL
PLSDLLLEM (SEQ ID NO:

QKYYITGE (SEQ ID NO: 32)
256)(A02.01)

PLSDLLLEML (SEQ ID

NO: 257)(A02.01)

VPLSDLLLEM (SEQ ID

NO: 258)(B07.02)

VVPLSDLLL (SEQ ID NO:

259)(A02.01, A24.02)

FGFR3
S249C
HRIGGIKLRHQQWSLVMES
VLERCPHRPI (SEQ ID NO:
BLCA, HNSC,

VVPSDRGNYTCVVENKFGS
260)(A02.01, B08.01)
KIRP, LUSC

IRQTYTLDVLERCPHRPILQ
YTLDVLERC (SEQ ID NO:

AGLPANQTAVLGSDVEFHC
261)(A02.01)

KVYSDAQPHIQWLKHVEV

NGSKVG (SEQ ID NO: 33)

FRG1B
L52S
AVKLSDSRIALKSGYGKYL
FQNGKMALS (SEQ ID NO:
GBM, KIRP,

GINSDELVGHSDAIGPREQ
262)(A02.01)
PRAD, SKCM

WEPVFQNGKMALSASNSC

FIRCNEAGDIEAKSKTAGEE

EMIKIRSCAEKETKKKDDIP

EEDKG (SEQ ID NO: 34)

HER2
V777L
GSGAFGTVYKGIWIPDGEN
VMAGLGSPYV (SEQ ID
BRCA

(Resistance)
VKIPVAIKVLRENTSPKAN
NO: 263)(A02.01, A03.01)

KEILDEAYVMAGLGSPYVS

RLLGICLTSTVQLVTQLMP

YGCLLDHVRENRGRLGSQ

DLLNWCM (SEQ ID NO: 35)

IDH1
R132H
RVEEFKLKQMWKSPNGTIR
KPIIIGHHA (SEQ ID NO:
BLCA, GBM,

NILGGTVFREAIICKNIPRLV
264)(B07.02)
PRAD

SGWVKPIIIGHHAYGDQYR

ATDFVVPGPGKVEITYTPS

DGTQKVTYLVHNFEEGGG

VAMGM (SEQ ID NO: 36)

IDH1
R132C
RVEEFKLKQMWKSPNGTIR
KPIIIGCHA (SEQ ID NO:
BLCA, GBM,

NILGGTVFREAIICKNIPRLV
265)(B07.02)
PRAD

SGWVKPIIIGCHAYGDQYR

ATDFVVPGPGKVEITYTPS

DGTQKVTYLVHNFEEGGG

VAMGM (SEQ ID NO: 37)

IDH1
R132G
RVEEFKLKQMWKSPNGTIR
KPIIIGGHA (SEQ ID NO:
BLCA, BRCA,

NILGGTVFREAIICKNIPRLV
266)(B07.02)
CRC, GBM,

SGWVKPIIIGGHAYGDQYR

HNSC, LUAD,

ATDFVVPGPGKVEITYTPS

PAAD, PRAD,

DGTQKVTYLVHNFEEGGG

UCEC

VAMGM (SEQ ID NO: 38)

IDH1
R132S
RVEEFKLKQMWKSPNGTIR
KPIIIGSHA (SEQ ID NO:
BLCA, BRCA,

NILGGTVFREAIICKNIPRLV
267)(B07.02)
GBM, HNSC,

SGWVKPIIIGSHAYGDQYR

LIHC, LUAD,

ATDFVVPGPGKVEITYTPS

LUSC, PAAD,

DGTQKVTYLVHNFEEGGG

SKCM, UCEC

VAMGM (SEQ ID NO: 39)

KIT
T670I
VAVKMLKPSAHLTEREAL
IIEYCCYGDL (SEQ ID NO:
Gastrointestinal

MSELKVLSYLGNHMNIVN
268)(A02.01)
stromal tumors

LLGACTIGGPTLVIIEYCCY
TIGGPTLVII (SEQ ID NO:
(GIST)

GDLLNFLRRKRDSFICSKQE
269)(A02.01)

DHAEAALYKNLLHSKESSC
VIIEYCCYG (SEQ ID NO:

SDSTNE (SEQ ID NO: 40)
270)(A02.01)

KIT
V654A
VEATAYGLIKSDAAMTVA
HMNIANLLGA (SEQ ID
Gastrointestinal

VKMLKPSAHLTEREALMSE
NO: 271)(A02.01)
stromal tumors

LKVLSYLGNHMNIANLLG
IANLLGACTI (SEQ ID NO:
(GIST)

ACTIGGPTLVITEYCCYGDL
272)(A02.01)

LNFLRRKRDSFICSKQEDH
MNIANLLGA (SEQ ID NO:

AEAALYK (SEQ ID NO: 41)
273)(A02.01)

YLGNHMNIA (SEQ ID NO:

274)(A02.01, B08.01)

YLGNHMNIAN (SEQ ID

NO: 275)(A02.01)

MEK
C121S
ISELGAGNGGVVFKVSHKP
VLHESNSPY (SEQ ID NO:
Melanoma

SGLVMARKLIHLEIKPAIRN
276)(A03.01)

QIIRELQVLHESNSPYIVGF
VLHESNSPYI (SEQ ID NO:

YGAFYSDGEISICMEHMDG
277)(A02.01)

GSLDQVLKKAGRIPEQILG

KVSI (SEQ ID NO: 42)

MEK
P124L
LGAGNGGVVFKVSHKPSG
LQVLHECNSL (SEQ ID
Melanoma

LVMARKLIHLEIKPAIRNQII
NO: 278)(A02.01, B08.01)

RELQVLHECNSLYIVGFYG
LYIVGFYGAF (SEQ ID

AFYSDGEISICMEHMDGGS
NO: 279)(A24.02)

LDQVLKKAGRIPEQILGKV
NSLYIVGFY (SEQ ID NO:

SIAVI (SEQ ID NO: 43)
280)(A01.01)

QVLHECNSL (SEQ ID NO:

281)(A02.01, B08.01)

SLYIVGFYG (SEQ ID NO:

282)(A02.01)

SLYIVGFYGA (SEQ ID

NO: 283)(A02.01)

VLHECNSLY (SEQ ID NO:

284)(A03.01)

VLHECNSLYI (SEQ ID

NO: 285)(A02.01, A03.01)

MYC
E39D
MPLNVSFTNRNYDLDYDS
FYQQQQQSDL (SEQ ID
Lymphoid

VQPYFYCDEEENFYQQQQ
NO: 286)(A24.02)
Cancer; Burkitt

QSDLQPPAPSEDIWKKFELL
QQQSDLQPPA (SEQ ID
Lymphoma

PTPPLSPSRRSGLCSPSYVA
NO: 287)(A02.01)

VTPFSLRGDNDGG (SEQ ID
QQSDLQPPA (SEQ ID NO:

NO: 44)
288)(A02.01)

YQQQQQSDL (SEQ ID NO:

289)(A02.01, B08.01)

MYC
P57S
FTNRNYDLDYDSVQPYFYC
FELLSTPPL (SEQ ID NO:
Lymphoid

DEEENFYQQQQQSELQPPA
290)(A02.01, B08.01)
Cancer

PSEDIWKKFELLSTPPLSPS
LLSTPPLSPS (SEQ ID NO:

RRSGLCSPSYVAVTPFSLRG
291)(A02.01)

DNDGGGGSFSTADQLEMV

TELLG (SEQ ID NO: 45)

MYC
T58I
TNRNYDLDYDSVQPYFYC
FELLPIPPL (SEQ ID NO:
Neuroblastoma

DEEENFYQQQQQSELQPPA
292)(A02.01)

PSEDIWKKFELLPIPPLSPSR
IWKKFELLPI (SEQ ID NO:

RSGLCSPSYVAVTPFSLRG
293)(A24.02)

DNDGGGGSFSTADQLEMV
LLPIPPLSPS (SEQ ID NO:

TELLGG (SEQ ID NO: 46)
294)(A02.01, B07.02)

LPIPPLSPS (SEQ ID NO:

295)(B07.02)

PDGFRa
T674I
VAVKMLKPTARSSEKQAL
IIEYCFYGDL (SEQ ID NO:
Chronic

MSELKIMTHLGPHLNIVNL
296)(A02.01)
Eosinophilic

LGACTKSGPIYIIIEYCFYGD
IIIEYCFYG (SEQ ID NO:
Leukemia

LVNYLHKNRDSFLSHHPEK
297)(A02.01)

PKKELDIFGLNPADESTRSY
IYIIIEYCF (SEQ ID NO:

VILS (SEQ ID NO: 47)
298)(A24.02)

IYIIIEYCFY (SEQ ID NO:

299)(A24.02)

YIIIEYCFYG (SEQ ID NO:

300)(A02.01)

PIK3CA
E542K
IEEHANWSVSREAGFSYSH
KITEQEKDFL (SEQ ID NO:
BLCA, BRCA,

AGLSNRLARDNELRENDKE
301)(A02.01)
CESC, CRC,

QLKAISTRDPLSKITEQEKD

GBM, HNSC,

FLWSHRHYCVTIPEILPKLL

KIRC, KIRP,

LSVKWNSRDEVAQMYCLV

LIHC, LUAD,

KDWPP (SEQ ID NO: 48)

LUSC, PRAD,

UCEC

PIK3CA
E545K
HANWSVSREAGFSYSHAG
STRDPLSEITK (SEQ ID
BLCA, BRCA,

LSNRLARDNELRENDKEQL
NO: 302)(A03.01)
CESC, CRC,

KAISTRDPLSEITKQEKDFL
DPLSEITK (SEQ ID NO:
GBM, HNSC,

WSHRHYCVTIPEILPKLLLS
303)(A03.01)
KIRC, KIRP,

VKWNSRDEVAQMYCLVK

LIHC, LUAD,

DWPPIKP (SEQ ID NO: 49)

LUSC, PRAD,

SKCM, UCEC

PIK3CA
H1047R
LFINLFSMMLGSGMPELQS

BRCA, CESC,

FDDIAYIRKTLALDKTEQE

CRC, GBM,

ALEYFMKQMNDARHGGW

HNSC, LIHC,

TTKMDWIFHTIKQHALN

LUAD, LUSC,

(SEQ ID NO: 50)

PRAD, UCEC

POLE
P286R
QRGGVITDEEETSKKIADQ
LPLKFRDAET (SEQ ID
Colorectal

LDNIVDMREYDVPYHIRLSI
NO: 304)(B07.02)
adenocarcinoma,

DIETTKLPLKFRDAETDQIM

Uterine/

MISYMIDGQGYLITNREIVS

Endometrium

EDIEDFEFTPKPEYEGPFCV

Adenocarcinoma;

FN (SEQ ID NO: 51)

Colorectal

adenocarcinoma,

MSI+;

Uterine/

Endometrium

Adenocarcinoma,

MSI+;

Endometrioid

carcinoma;

Endometrium

Serous

carcinoma;

Endometrium

Carcinosarcoma-

malignant

mesodermal

mixed tumor;

Glioma;

Astrocytoma;

GBM

PTEN
R130Q
KFNCRVAQYPFEDHNPPQL
QTGVMICAYL (SEQ ID
BRCA, CESC,

ELIKPFCEDLDQWLSEDDN
NO: 305)(A02.01)
CRC, GBM,

HVAAIHCKAGKGQTGVMI

KIRC, LUSC,

CAYLLHRGKFLKAQEALDF

UCEC

YGEVRTRDKKGVTIPSQRR

YVYYYSY (SEQ ID NO: 52)

RAC1
P29S
MQAIKCVVVGDGAVGKTC
AFSGEYIPTV (SEQ ID NO:
Melanoma

LLISYTTNAFSGEYIPTVFD
306)(A02.01, A24.02)

NYSANVMVDGKPVNLGL

WDTAGQEDYDRLRPLSYP

QTVGET (SEQ ID NO: 53)

TP53
G245S
IRVEGNLRVEYLDDRNTFR
SMNRRPILT (SEQ ID NO:
BLCA, BRCA,

HSVVVPYEPPEVGSDCTTIH
307)(A02.01, B08.01)
CRC, GBM,

YNYMCNSSCMGSMNRRPI
YMCNSSCMGS (SEQ ID
HNSC, LUSC,

LTIITLEDSSGNLLGRNSFE
NO: 308)(A02.01)
PAAD, PRAD

VRVCACPGRDRRTEEENLR

KKGEP (SEQ ID NO: 54)

TP53
R175H
TYSPALNKMFCQLAKTCPV

BLCA, BRCA,

QLWVDSTPPPGTRVRAMAI

CRC, GBM,

YKQSQHMTEVVRHCPHHE

HNSC, LUAD,

RCSDSDGLAPPQHLIRVEG

PAAD, PRAD,

NLRVEYLDDRNTFRHSVV

UCEC

VPYEPPEV (SEQ ID NO: 55)

TP53
R248Q
EGNLRVEYLDDRNTFRHSV
GMNQRPILT (SEQ ID NO:
BLCA, BRCA,

VVPYEPPEVGSDCTTIHYN
309)(A02.01)
CRC, GBM,

YMCNSSCMGGMNQRPILTI

HNSC, KIRC,

ITLEDSSGNLLGRNSFEVRV

LIHC, LUSC,

CACPGRDRRTEEENLRKKG

PAAD, PRAD,

EPHHE (SEQ ID NO: 56)

UCEC

TP53
R248W
EGNLRVEYLDDRNTFRHSV
GMNWRPILT (SEQ ID NO:
BLCA, BRCA,

VVPYEPPEVGSDCTTIHYN
310)(A02.01)
CRC, GBM,

YMCNSSCMGGMNWRPILT

HNSC, LIHC,

IITLEDSSGNLLGRNSFEVR

LUSC, PAAD,

VCACPGRDRRTEEENLRKK

SKCM, UCEC

GEPHHE (SEQ ID NO: 57)

TP53
R273C
PEVGSDCTTIHYNYMCNSS
LLGRNSFEVC (SEQ ID
BLCA, BRCA,

CMGGMNRRPILTIITLEDSS
NO. 311)(A02.01)
CRC, GBM,

GNLLGRNSFEVCVCACPGR

HNSC, LUSC,

DRRTEEENLRKKGEPHHEL

PAAD, UCEC

PPGSTKRALPNNTSSSPQPK

KKPL (SEQ ID NO: 58)

TABLE 1B

MSI-ASSOCIATED FRAMESHIFTS

ACVR2A
D96fs;
GVEPCYGDKDKRRHCFAT

MSI+CRC,

+1
WKNISGSIEIVKQGCWLDDI

MSI+

NCYDRTDCVEKKRQP*

Uterine/Endo-

(SEQ ID NO: 59)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

ACVR2A
D96fs;
GVEPCYGDKDKRRHCFAT
ALKYIFVAV (SEQ ID NO:
MSI+ CRC,

−1
WKNISGSIEIVKQGCWLDDI
312) (A02.01, B08.01)
MSI+

NCYDRTDCVEKKTALKYIF
ALKYIFVAVR (SEQ ID
Uterine/Endo-

VAVRAICVMKSFLIFRRWK

NO: 313) (A03.01)
metrium Cancer,

SHSPLQIQLHLSHPITTSCSI

AVRAICVMK (SEQ ID NO:
MST+ Stomach

PWCHLC* (SEQ ID NO: 60)
314) (A03.01)
Cancer, Lynch

AVRAICVMKS (SEQ ID
syndrome

NO: 315) (A03.01)

CVEKKTALK (SEQ ID NO:

316) (A03.01)

CVEKKTALKY (SEQ ID

NO: 317) (A01.01)

CVMKSFLIF (SEQ ID NO:

318) (A24.02, B08.01)

CVMKSFLIFR (SEQ ID

NO: 319) (A03.01)

FLIFRRWKS (SEQ ID NO:

320) (A02.01, B08.01)

FRRWKSHSPL (SEQ ID

NO: 321) (B08.01)

FVAVRAICV (SEQ ID NO:

322) (A02.01, B08.01)

FVAVRAICVM (SEQ ID

NO: 323) (B08.01)

IQLEILSHPI (SEQ ID NO:

324) (A02.01)

KSFLIFRRWK (SEQ ID

NO: 325) (A03.01)

KTALKYIFV (SEQ ID NO:

326) (A02.01)

KYIFVAVRAI (SEQ ID NO:

327) (A24.02)

RWKSHSPLQI (SEQ ID

NO: 328) (A24.02)

TALKYIFVAV (SEQ ID

NO: 329) (A02.01, B08.01)

VAVRAICVMK (SEQ ID

NO: 330) (A03.01)

VMKSFLIFR (SEQ ID NO:

331) (A03.01)

VMKSFLIFRR (SEQ ID

NO: 332) (A03.01)

YIFVAVRAI (SEQ ID NO:

333) (A02.01)

C15ORF40
L132fs;
TAEAVNVAIAAPPSEGEAN
ALFFFFFET (SEQ ID NO:
MSI+ CRC,

+1
AELCRYLSKVLELRKSDVV
334) (A02.01)
MSI+

LDKVGLALFFFFFETKSCSV
ALFFFFFETK (SEQ ID NO:
Uterine/Endo-

AQAGVQWRSLGSLQPPPPG

335) (A03.01)
metrium Cancer,

FKLFSCLSFLSSWDYRRMP

AQAGVQWRSL (SEQ ID
MSI+ Stomach

PCLANFCIFNRDGVSPCWS

NO: 336) (A02.01)
Cancer, Lynch

GWS* (SEQ ID NO: 61)
CLANFCIFNR (SEQ ID NO:
syndrome

337) (A03.01)

CLSFLSSWDY (SEQ ID

NO: 338) (A01.01, A03.01)

FFETKSCSV (SEQ ID NO:

339) (B08.01)

FFFETKSCSV (SEQ ID NO:

340) (A02.01)

FKLFSCLSFL (SEQ ID NO:

341) (A02.01)

FLSSWDYRRM (SEQ ID

NO. 342) (A02.01)

GFKLFSCLSF (SEQ ID NO:

343) (A24.02)

KLFSCLSFL (SEQ ID NO:

344) (A02.01, A03.01)

KLFSCLSFLS (SEQ ID NO:

345) (A02.01, A03.01)

LALFFFFFET (SEQ ID NO:

346) (A02.01)

LFFFFFETK (SEQ ID NO:

347) (A03.01)

LSFLSSWDY (SEQ ID NO:

348) (A01.01)

LSFLSSWDYR (SEQ ID

NO: 349) (A03.01)

RMPPCLANF (SEQ ID NO:

350) (A24.02)

RRMPPCLANF (SEQ ID

NO: 351) (A24.02)

SLQPPPPGFK (SEQ ID NO:

352) (A03.01)

VQWRSLGSL (SEQ ID NO:

353) (A02.01)

CNOT1
L1544fs;
LSVIIFFFVYIWHWALPLIL
FFFSVIFST (SEQ ID NO:
MSI+ CRC,

+1
NNHHICLMSSIILDCNSVRQ
354) (A02.01)
MSI+

SIMSVCFFFFSVIFSTRCLTD
MSVCFFFFSV (SEQ ID
Uterine/Endo-

SRYPNICWFK* (SEQ ID NO:
NO: 355) (A02.01)
metrium Cancer,

62)
SVCFFFFSV (SEQ ID NO:
MSI+ Stomach

356) (A02.01, B08.01)
Cancer, Lynch

SVCFFFFSVI (SEQ ID NO:
syndrome

357) (A02.01)

CNOT1
L1544fs;
LSVIIFFFVYIWHWALPLIL
FFCYILNTMF (SEQ ID NO:
MSI+ CRC

−1
NNHHICLMSSIILDCNSVRQ
358) (A24.02)
MSI+

SIMSVCFFFFCYILNTMFDR*
MSVCFFFFCY (SEQ ID
Uterine/Endo-

(SEQ ID NO: 63)
NO: 359) (A01.01)
metrium Cancer,

SVCFFFFCYI (SEQ ID NO:
MSI+ Stomach

360) (A02.01)
Cancer, Lynch

syndrome

EIF2B3
A151fs;
VLVLSCDLITDVALHEVVD
KQWSSVTSL (SEQ ID NO:
MSI+ CRC,

−1
LFRAYDASLAMLMRKGQD
361) (A02. 01)
MSI+

SIEPVPGQKGKKKQWSSVT
VLWMPTSTV (SEQ ID NO:
Uterine/Endo-

SLEWTAQERGCSSWLMKQ

362) (A02.01)
metrium Cancer,

TWMKSWSLRDPSYRSILEY

MSI+ Stomach

VSTRVLWMPTSTV* (SEQ

Cancer, Lynch

ID NO: 64)

syndrome

EPHB2
K1020fs;
SIQVMRAQMNQIQSVEGQP
ILIRKAMTV (SEQ ID
MSI+ CRC,

−1
LARRPRATGRTKRCQPRDV
NO. 363) (A02.01)
MSI+

TKKTCNSNDGKKREWEKR

Uterine/Endo-

KQILGGGGKYKEYFLKRILI

metrium Cancer,

RKAMTVLAGDKKGLGRFM

MSI+ Stomach

RCVQSETKAVSLQLPLGR*

Cancer, Lynch

(SEQ ID NO: 65)

syndrome

ESRP1
N512fs;
LDFLGEFATDIRTHGVHMV

MSI+ CRC,

+1
LNHQGRPSGDAFIQMKSAD

MSI+

RAFMAAQKCHKKKHEGQI

Uterine/Endo-

C* (SEQ ID NO: 66)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

ESRP1
N512fs;
LDFLGEFATDIRTHGVHMV

MSI+ CRC,

−1
LNHQGRPSGDAFIQMKSAD

MSI+

RAFMAAQKCHKKT* (SEQ

Uterine/Endo-

ID NO: 67)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

FAM111B
A273fs;
GALCKDGRFRSDIGEFEWK
RMKVPLMK (SEQ ID NO:
MSI+ CRC,

−1
LKEGHKKIYGKQSMVDEV
364) (A03.01)
MSI+

SGKVLEMDISKKKHYNRKI

Uterine/Endo-

SIKKLNRMKVPLMKLITRV*

metrium Cancer,

(SEQ ID NO: 68)

MSI+ Stomach

Cancer, Lynch

syndrome

GBP3
T585fs;
RERAQLLEEQEKTLTSKLQ
TLKKKPRDI (SEQ ID
MSI+ CRC,

−1
EQARVLKERCQGESTQLQN
NO: 365) (B08.01)
MSI+

EIQKLQKTLKKKPRDICRIS*

Uterine/Endo-

(SEQ ID NO: 69)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

JAK1
P861fs;
VNTLKEGKRLPCPPNCPDE
LIEGFEALLK (SEQ ID NO:
MSI+ CRC,

+1
VYQLMRKCWEFQPSNRTS
366) (A03.01)
MSI+

FQNLIEGFEALLKTSN*

Uterine/Endo-

(SEQ ID NO: 70)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

JAK1
K860fs;
CRPVTPSCKELADLMTRCM
QQLKWTPHI (SEQ ID NO:
MSI+ CRC,

−1
NYDPNQRPFFRAIMRDINK
367) (A02.01)
MSI+

LEEQNPDIVSEKNQQLKWT
QLKWTPHILK (SEQ ID
Uterine/Endo-

PHILKSAS*

NO: 368) (A03.01)
metrium Cancer,

(SEQ ID NO: 71)
IVSEKNQQLK (SEQ ID
MSI+ Stomach

NO: 369) (A03.01)
Cancer, Lynch

QLKWTPHILK (SEQ ID
syndrome

NO: 368) (A03.01)

QQLKWTPHI (SEQ ID NO:

367) (A24.02)

NQQLKWTPHIL (SEQ ID

NO: 370) (B08.01)

NQQLKWTPHI (SEQ ID

NO: 371) (B08.01)

QLKWTPHIL (SEQ ID NO:

372) (B08.01)

LMAN1
E305fs;
DDHDVLSFLTFQLTEPGKE
GPPRPPRAAC (SEQ ID
MSI+ CRC,

+1
PPTPDKEISEKEKEKYQEEF
NO: 373) (B07.02)
MSI+

EHFQQELDKKKRGIPEGPP
PPRPPRAAC (SEQ ID NO:
Uterine/Endo-

RPPRAACGGNI* (SEQ ID
374) (B07.02)
metrium Cancer,

NO: 72)

MSI+ Stomach

Cancer, Lynch

syndrome

LMAN1
E305fs;
DDHDVLSFLTFQLTEPGKE
SLRRKYLRV (SEQ ID NO:
MSI+ CRC,

−1
PTPDKEISEKEKEKYQEEF
375) (B08.01)
MSI+

EHFQQELDKKKRNSRRATP

Uterine/Endo-

TSKGSLRRKYLRV* (SEQ

metrium Cancer,

ID NO: 73)

MSI+ Stomach

Cancer, Lynch

syndrome

MSH3
N385fs;
TKSTLIGEDVNPLIKLDDAV
SAACHRRGCV (SEQ ID
MSI+ CRC,

+1
NVDEIMTDTSTSYLLCISEN
NO: 376) (B08.01)
MSI+

KENVRDKKKGQHFYWHC

Uterine/Endo-

GSAACHRRGCV* (SEQ ID

metrium Cancer,

NO: 74)

MSI+ Stomach

Cancer, Lynch

syndrome

MSH3
K383fs;
LYTKSTLIGEDVNPLIKLDD
ALWECSLPQA (SEQ ID
MSI+ CRC,

−1
AVNVDEIMTDTSTSYLLCIS
NO: 377) (A02.01)
MSI+

ENKENVRDKKRATFLLAL
CLIVSRTLL (SEQ ID NO:
Uterine/Endo-

WECSLPQARLCLIVSRTLLL

378) (B08.01)
metrium Cancer,

VQS* (SEQ ID NO: 75)
CLIVSRTLLL (SEQ ID NO:
MSI+ Stomach

379) (A02.01, B08.01)
Cancer, Lynch

FLLALWECS (SEQ ID NO:
syndrome

380) (A02.01)

FLLALWECSL (SEQ ID

NO: 381) (A02.01, B08.01)

IVSRTLLLV (SEQ ID NO:

382) (A02.01)

LIVSRTLLL (SEQ ID NO:

383) (A02.01, B08.01)

LIVSRTLLLV (SEQ ID NO:

384) (A02.01)

LLALWECSL (SEQ ID NO:

385) (A02.01, B08.01)

LPQARLCLI (SEQ ID NO:

386) (B08.01, B07.02)

LPQARLCLIV (SEQ ID NO:

387) (B08.01)

NVRDKKRATF (SEQ ID

NO: 388) (B08.01)

SLPQARLCLI (SEQ ID NO:

389) (A02.01, B08.01)

NDUFC2
A70fs;
LPPPKLTDPRLLYIGFLGYC
FFCWILSCK (SEQ ID NO:
MSI+ CRC,

+1
SGLIDNLIRRRPIATAGLEIR
390) (A03.01)
MSI+

QLLYITAFFFCWILSCKT*
FFFCWILSCK (SEQ ID NO:
Uterine/Endo-

(SEQ ID NO: 76)
391) (A03.01)
metrium Cancer,

ITAFFFCWI (SEQ ID NO:
MSI+ Stomach

392) (A02.01)
Cancer, Lynch

LYITAFFFCW (SEQ ID
syndrome

NO: 393) (A24.02)

YITAFFFCWI (SEQ ID NO:

394) (A02.01)

NDUFC2
F69fs;
SLPPPKLTDPRLLYIGFLGY
ITAFFLLDI (SEQ ID NO:
MSI+ CRC,

−1
CSGLIDNLIRRRPIATAGLH
395) (A02.01)
MSI+

RQLLYITAFFLLDIIL* (SEQ
LLYITAFFL (SEQ ID NO:
Uterine/Endo-

ID NO: 77)
396) (A02.01, B08.01)
metrium Cancer,

LLYITAFFLL (SEQ ID NO:
MSI+ Stomach

397) (A02.01, A24.02)
Cancer, Lynch

LYITAFFLL (SEQ ID NO:
syndrome

398) (A24.02)

LYITAFFLLD (SEQ ID NO:

399) (A24.02)

YITAFFLLDI (SEQ ID NO:

400) (A02.01)

RBM27
Q817;
NQSGGAGEDCQIFSTPGHP
GSNEVTTRY (SEQ ID NO:
MSI+ CRC,

+1
KMIYSSSNLKTPSKLCSGSK
401) (A01.01)
MSI+

SHDVQEVLKKKTGSNEVTT
MPKDVNIQV (SEQ ID NO:
Uterine/Endo-

RYEEKKTGSVRKANRMPK

402) (B07.02)
metrium Cancer,

DVNIQVRKKQKHETRRKS

TGSNEVTTRY (SEQ ID
MSI+ Stomach

KYNEDFERAWREDLTIKR*

NO: 403) (A01.01)
Cancer, Lynch

(SEQ ID NO: 78)

syndrome

RPL22
K16fs;
MAPVKKLVVKGGKKKEAS

MSI+ CRC,

+1

SEVHS* (SEQ ID NO: 79)

MSI+

Uterine/Endo-

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

RPL22
K15fs;
MAPVKKLVVKGGKKRSKF*

MSI+ CRC,

−1
(SEQ ID NO: 80)

MSI+

Uterine/Endo-

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

SEC31A
I462fs;
MPSHQGAEQQQQQHHVFIS

MSI+ CRC,

+1
QVVTEKEFLSRSDQLQQAV

MSI+

QSQGFINYCQKKN* (SEQ

Uterine/Endo-

ID NO: 81)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

SEC31A
I462fs;
MPSHQGAEQQQQQHHVFIS
KKLMLLRLNL (SEQ ID
MSI+ CRC,

−1
QVVTEKEFLSRSDQLQQAV
NO: 404) (A02.01)
MSI+

QSQGFINYCQKKLMLLRLN
KLMLLRLNL (SEQ ID NO:
Uterine/Endo-

LRKMCGPF* (SEQ ID NO:
405) (A02.01, A03.01,
metrium Cancer,

82)
B07.02, B08.01)
MSI+ Stomach

KLMLLRLNLR (SEQ ID
Cancer, Lynch

NO: 406) (A03.01)
syndrome

LLRLNLRKM (SEQ ID NO:

407) (B08.01)

LMLLRLNL (SEQ ID NO:

408) (B08.01)

LMLLRLNLRK (SEQ ID

NO: 409) (A03.01)

LNLCGPF (SEQ ID

NO: 410) (B08.01)

MLLRLNLRK (SEQ ID NO:

411) (A03.01)

MLLRLNLRKM (SEQ ID

NO: 412) (A02.01, A03.01,

B08.01)

NLRKMCGPF (SEQ ID NO:

413) (B08.01)

NYCQKKLMLL (SEQ ID

NO: 414) (A24.02)

YCQKKLMLL (SEQ ID

NO: 415) (B08.01)

SEC63
K530fs;
AEVFEKEQSICAAEEQPAE
FKKKTYTCAI (SEQ ID
MSI+ CRC,

+1
DGQGETNKNRTKGGWQQ
NO: 416) (B08.01)
MSI+

KSKGPKKTAKSKKKETFKK
ITTVKATETK (SEQ ID
Uterine/Endo-

KTYTCAITTVKATETKAGK

NO: 417) (A03.01)
metrium Cancer,

WSRWE* (SEQ ID NO: 83)
KSKKKETFK (SEQ ID NO:
MSI+ Stomach

418) (A03.01)
Cancer, Lynch

KSKKKETFKK (SEQ ID
syndrome

NO: 419) (A03.01)

KTYTCAITTV (SEQ ID

NO: 420) (A02.01, A24.02)

TFKKKTYTC (SEQ ID NO:

421) (B08.01)

TYTCAITTV (SEQ ID NO:

422) (A24.02)

TYTCAITTVK (SEQ ID

NO: 423) (A03.01)

YTCAITTVK (SEQ ID NO:

424) (A03.01)

SEC63
K529fs;
MAEVFEKEQSICAAEEQPA
TAKSKKRNL (SEQ ID NO:
MSI+ CRC,

−1
EDGQGETNKNRTKGGWQQ
425) (B08.01)
MSI+

KSKGPKKTAKSKKRNL*

Uterine/Endo-

(SEQ ID NO: 84)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

SLC35F5
C248fs;
NIMEIRQLPSSHALEAKLSR
FALCGFWQI (SEQ ID NO:
MSI+ CRC,

−1
MSYPVKEQESILKTVGKLT
426) (A02.01)
MSI+

ATQVAKISFFFALCGFWQIC

Uterine/Endo-

HIKKHFQTEIKLL* (SEQ ID

metrium Cancer,

NO: 85)

MSI+ Stomach

Cancer, Lynch

syndrome

SMAP1
K172fs;
YEKKKYYDKNAIAITNISSS

MSI+ CRC,

+1
DAPLQPLVSSPSLQAAVDK

MSI+

NKLEKEKEKKKGREKERK

Uterine/Endo-

GARKAGKTTYS* (SEQ ID

metrium Cancer,

NO: 86)

MSI+ Stomach

Cancer, Lynch

syndrome

SMAP1
K171fs;
KYEKKKYYDKNAIAITNISS
LKKLRSPL (SEQ ID NO:
MSI+ CRC,

−1
SDAPLQPLVSSPSLQAAVD
427) (B08.01)
MSI+

KNKLEKEKEKKRKRKREK
SLKKVPAL (SEQ ID NO:
Uterine/Endo-

RSQKSRQNEILQLKSCRRKI

428) (B08.01)
metrium Cancer,

SNWSLKKVPALKKLRSPL

RKISNWSLKK (SEQ ID
MSI+ Stomach

WIF* (SEQ ID NO: 87)
NO: 429) (A03.01)
Cancer, Lynch

VPALKKLRSPL (SEQ ID
syndrome

NO: 430) (B07.02)

TFAM
E148fs;
IYQDAYRAEWQVYKEEISR
KRVNTAWKTK (SEQ ID
MSI+ CRC,

+1
FKEQLTPSQIMSLEKEIMDK
NO: 431) (A03.01)
MSI+

HLKRKAMTKKKRVNTAW
MTKKKRVNTA (SEQ ID
Uterine/Endo-

KTKKTSFSL*

NO: 432) (B08.01)
metrium Cancer,

(SEQ ID NO: 88)
RVNTAWKTK (SEQ ID
MSI+ Stomach

NO: 433) (A03.01)
Cancer, Lynch

RVNTAWKTKK (SEQ ID
syndrome

NO: 434) (A03.01)

TKKKRVNTA (SEQ ID NO:

435) (B08.01)

WKTKKTSFSL (SEQ ID

NO: 436) (B08.01)

TFAM
E148fs;
IYQDAYRAEWQVYKEEISR

MSI+CRC,

−1
FKEQLTPSQIMSLEKEIMDK

MSI+

HLKRKAMTKKKS* (SEQ ID

Uterine/Endo-

NO: 89)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

TGFBR2
P129fs;
KPQEVCVAVWRKNDENIT

MSI+ CRC,

+1
LETVCHDPKLPYHDFILED

MSI+

AASPKCIMKEKKKAW*

Uterine/Endo-

(SEQ ID NO: 90)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

TGFBR2
K128fs;
EKPQEVCVAVWRKNDENI
ALMSAMTTS (SEQ ID NO:
MSI+ CRC,

−1
TLETVCHDPKLPYHDFILED
437) (A02.01)
MSI+

AASPKCIMKEKKSLVRLSS
AMTTSSSQK (SEQ ID NO:
Uterine/Endo-

CVPVALMSAMTTSSSQKNI

438) (A03.01, A11.01)
metrium Cancer,

TPAILTCC*

AMTTSSSQKN (SEQ ID
MSI+ Stomach

(SEQ ID NO: 91)
NO: 439) (A03.01)
Cancer, Lynch

CIMKEKKSL (SEQ ID NO:
syndrome

440) (B08.01)

CIMKEKKSLV (SEQ ID

NO: 441) (B08.01)

IMKEKKSL (SEQ ID NO:

442) (B08.01)

IMKEKKSLV (SEQ ID NO:

443) (B08.01)

KSLVRLSSCV (SEQ ID

NO: 444) (A02.01)

LVRLSSCVPV (SEQ ID

NO: 445) (A02.01)

RLSSCVPVA (SEQ ID NO:

446) (A02.01, A03.01)

RLSSCVPVAL (SEQ ID

NO: 447) (A02.01)

SAMTTSSSQK (SEQ ID

NO: 448) (A03.01, A11.01)

SLVRLSSCV (SEQ ID NO:

449) (A02.01)

VPVALMSAM (SEQ ID

NO: 450) (B07.02)

VRLSSCVPVA (SEQ ID

NO: 451) (A02.01)

THAP5
K99fs;
VPSKYQFLCSDEIFTPDSLDI
KMRKKYAQK (SEQ ID
MSI+ CRC,

−1
RWGIRYLKQTAVPTIFSLPE
NO: 452) (A03.01)
MSI+

DNQGKDPSKKNPRRKTWK

Uterine/Endo-

MRKKYAQKPSQKNHLY*

metrium Cancer,

(SEQ ID NO: 92)

MSI+ Stomach

Cancer, Lynch

syndrome

TTK
R854fs;
GTTEEMKYVLGQLVGLNS
FVMSDTTYK (SEQ ID NO:
MSI+ CRC,

−1
PNSILKAAKTLYEHYSGGE
453) (A03.01)
MSI+

SHNSSSSKTFEKKGEKNDL
FVMSDTTYKI (SEQ ID
Uterine/Endo-

QLFVMSDTTYKIYWTVILL

NO: 454) (A02.01)
metrium Cancer,

NPCGNLEILKTTSL*

KTFEKKGEK (SEQ ID NO:
MSI+ Stomach

(SEQ ID NO: 93)
455) (A03.01)
Cancer, Lynch

LFVMSDTTYK (SEQ ID
syndrome

NO: 456) (A03.01)

MSDTTYKIY (SEQ ID NO:

457) (A01.01)

VMSDTTYKI (SEQ ID NO:

458) (A02.01)

VMSDTTYKIY (SEQ ID

NO: 459) (A01.01)

XPOT
F126fs;
QQLIRETLISWLQAQMLNP
YLTKWPKFFL (SEQ ID
MSI+ CRC,

−1
QPEKTFIRNKAAQVFALLF
NO: 460) (A02.01)
MSI+

VTEYLTKWPKFFLTFSQ*

Uterine/Endo-

(SEQ ID NO: 94)

metrium Cancer,

MSI+ Stomach

Cancer, Lynch

syndrome

TABLE 1C

FRAMESHIFT

APC
V1352fs

AKFQQCHSTLEPNPADCRV

FLQERNLPP (SEQ ID NO:
CRC, LUAD,

F1354fs

LVYLQNQPGTKLLNFLQER

461)(A02.01)
UCEC, STAD

Q1378fs

NLPPKVVLRHPKVHLNTMF

FRRPHSCLA (SEQ ID NO:

S1398fs

RRPHSCLADVLLSVHLIVL

462)(B08. 01)

RVVRLPAPFRVNHAVEW*

LIVLRVVRL (SEQ ID NO:

(SEQ ID NO: 95)
463)(B08.01)

LLSVHLIVL (SEQ ID NO:

464)(A02.01, B08.01)

APC
S1421fs

APVIFQIALDKPCHQAEVK

EVKHLHHLL (SEQ ID NO:
CRC, LUAD,

R1435fs

HLHHLLKQLKPSEKYLKIK

465)(B08.01)
UCEC, STAD

T1438fs

HLLLKRERVDLSKLQ*

HLHHLLKQLK (SEQ ID

P1442fs
(SEQ ID NO: 96)
NO: 466)(A03.01)

P1443fs

HLLLKRERV (SEQ ID NO:

V1452fs

467)(B08.01)

P1453fs

KIKHLLLKR (SEQ ID NO:

K1462fs

468)(A03.01)

E1464fs

KPSEKYLKI (SEQ ID NO:

469)(B07.02)

KYLKIKHLL (SEQ ID NO:

470)(A24.02)

KYLKIKHLLL (SEQ ID

NO: 471)(A24.02)

LLKQLKPSEK (SEQ ID

NO: 472)(A03.01)

LLKRERVDL (SEQ ID NO:

473)(B08.01)

LLLKRERVDL (SEQ ID

NO: 474)(B08.01)

QLKPSEKYLK (SEQ ID

NO: 475)(A03.01)

YLKIKHLLL (SEQ ID NO:

476)(A02.01, B08.01)

YLKIKHLLLK (SEQ ID

NO: 477)(A03.01)

APC
T1487fs

MLQFRGSRFFQMLILYYILP

ILPRKVLQM (SEQ ID NO:
CRC, LUAD,

H1490fs

RKVLQMDFLVHPA* (SEQ
478)(B08.01)
UCEC, STAD

L1488fs
ID NO: 97)
KVLQMDFLV (SEQ ID

NO: 479)(A02.01, A24.02)

LPRKVLQMDF (SEQ ID

NO: 480)(B07.02, B08.01)

LQMDFLVHPA (SEQ ID

NO: 481)(A02.01)

QMDFLVHPA (SEQ ID NO:

482)(A02.01)

YILPRKVLQM (SEQ ID

NO: 483)(A02.01, B08.01)

ARID1
Q1306fs

ALGPHSRISCLPTQTRGCIL

APSPASRLQC (SEQ ID
STAD, UCEC,

A
S1316fs

LAATPRSSSSSSSNDMIPMA

NO: 484)(B07.02)
BLCA, BRCA,

Y1324fs

ISSPPKAPLLAAPSPASRLQ

HPLAPMPSKT (SEQ ID
LUSC, CESC,

T1348fs

CINSNSRITSGQWMAHMAL

NO: 485)(B07.02)
KIRC, UCS

G1351fs

LPSGTKGRCTACHTALGRG

ILPLPQLLL (SEQ ID NO:

G1378fs

SLSSSSCPQPSPSLPASNKLP

486)(A02.01)

P1467fs

SLPLSKMYTTSMAMPILPLP

LPTQTRGCIL (SEQ ID NO:

QLLLSADQQAAPRTNFHSS

487)(B07.02)

LAETVSLHPLAPMPSKTCH

RISCLPTQTR (SEQ ID NO:

HK* (SEQ ID NO: 98)
488)(A03.01)

SLAETVSLH (SEQ ID NO:

489)(A03.01)

TPRSSSSSS (SEQ ID NO:

490)(B07.02)

TPRSSSSSSS (SEQ ID NO:

491)(B07.02)

ARID1
S674fs

AHQGFPAAKESRVIQLSLLS

ALPPVLLSL (SEQ ID NO:
STAD, UCEC,

A
P725fs

LLIPPLTCLASEALPRPLLAL

492)(A02.01)
BLCA, BRCA,

R727fs

PPVLLSLAQDHSRLLQCQA

ALPPVLLSLA (SEQ ID
LUSC, CESC,

I736fs

TRCHLGHPVASRTASCILP*

NO: 493)(A02.01)
KIRC, UCS

(SEQ ID NO: 99)
ALPRPLLAL (SEQ ID NO:

494)(A02.01)

ASRTASCIL (SEQ ID NO:

495)(B07.02)

EALPRPLLAL (SEQ ID

NO: 496)(B08.01)

HLGHPVASR (SEQ ID NO:

497)(A03.01)

HPVASRTAS (SEQ ID NO:

498)(B07.02)

HPVASRTASC (SEQ ID

NO: 499)(B07.02)

IIQLSLLSLL (SEQ ID NO:

500)(A02.01)

IQLSLLSLL (SEQ ID NO:

501)(A02.01)

IQLSLLSLLI (SEQ ID NO:

502)(A02.01, A24.02)

LLALPPVLL (SEQ ID NO:

503)(A02.01)

LLIPPLTCL (SEQ ID NO:

504)(A02.01)

LLIPPLTCLA (SEQ ID NO:

505)(A02.01)

LLSLLIPPL (SEQ ID NO:

506)(A02.01)

LLSLLIPPLT (SEQ ID NO:

507)(A02.01)

LPRPLLALPP (SEQ ID NO:

508)(B07.02)

QLSLLSLLI (SEQ ID NO:

509)(A02.01)

RLLQCQATR (SEQ ID NO:

510)(A03.01)

RPLLALPPV (SEQ ID NO:

511)(B07.02)

RPLLALPPVL (SEQ ID NO:

512)(B07.02)

SLAQDHSRL (SEQ ID NO:

513)(A02.01)

SLAQDHSRLL (SEQ ID

NO: 514)(A02.01)

SLLIPPLTCL (SEQ ID NO:

515)(A02.01)

SLLSLLIPP (SEQ ID NO:

516)(A02.01)

SLLSLLIPPL (SEQ ID NO:

517)(A02.01, B08.01)

ARID1
G414fs

PILAATGTSVRTAARTWVP

AAATSAASTL (SEQ ID
STAD, UCEC,

A
Q473fs

RAAIRVPDPAAVPDDHAGP

NO: 518)(B07.02)
BLCA, BRCA,

H477fs

GAECHGRPLLYTADSSLWT

AAIPASTSAV (SEQ ID NO:
LUSC, CESC,

S499fs

TRPQRVWSTGPDSILQPAK

519)(B07.02)
KIRC, UCS

P504fs

SSPSAAAATLLPATTVPDPS

AIPASTSAV (SEQ ID NO:

Q548fs

CPTFVSAAATVSTTTAPVLS

520)(A02.01)

P549fs

ASILPAAIPASTSAVPGSIPL

ALPAGCVSSA (SEQ ID

PAVDDTAAPPEPAPLLTAT

NO: 521)(A02.01)

GSVSLPAAATSAASTLDAL

APLLTATGSV (SEQ ID

PAGCVSSAPVSAVPANCLF

NO: 522)(B07.02)

PAALPSTAGAISRFIWVSGI

APVLSASIL (SEQ ID NO:

LSPLNDLQ* (SEQ ID NO:
523)(B07.02)

100)
ATLLPATTV (SEQ ID NO:

524)(A02.01)

ATVSTTTAPV (SEQ ID

NO: 525)(A02.01)

AVPANCLFPA (SEQ ID

NO: 526)(A02.01)

CLFPAALPST (SEQ ID NO:

527)(A02.01)

CPTFVSAAA (SEQ ID NO:

528)(B07.02)

FPAALPSTA (SEQ ID NO:

529)(B07.02)

FPAALPSTAG (SEQ ID

NO. 530)(B07.02)

GAECHGRPL (SEQ ID NO:

531)(B07.02)

GAISRFIWV (SEQ ID NO:

532)(A02.01)

ILPAAIPAST (SEQ ID NO:

533)(A02.01)

IWVSGILSPL (SEQ ID NO:

534)(A24.02)

LLTATGSVSL (SEQ ID

NO: 535)(A02.01)

LLYTADSSL (SEQ ID NO:

536)(A02.01)

LPAAATSAA (SEQ ID NO:

537)(B07.02)

LPAAATSAAS (SEQ ID

NO: 538)(B07.02)

LPAAIPAST (SEQ ID NO:

539)(B07.02)

LPAGCVSSA (SEQ ID NO:

540)(B07.02)

LPAGCVSSAP (SEQ ID

NO: 541)(B07.02)

LYTADSSLW (SEQ ID NO:

542)(A24.02)

QPAKSSPSA (SEQ ID NO:

543)(B07.02)

QPAKSSPSAA (SEQ ID

NO: 544)(B07.02)

RFIWVSGIL (SEQ ID NO:

545)(A24.02)

RPQRVWSTG (SEQ ID NO:

546)(B07.02)

RVWSTGPDSI (SEQ ID

NO: 547)(A02.01)

SAVPGSIPL (SEQ ID NO:

548)(B07.02)

SILPAAIPA (SEQ ID NO:

549)(A02.01)

SLPAAATSA (SEQ ID NO:

550)(A02.01)

SLPAAATSAA (SEQ ID

NO: 551)(A02.01)

SLWTTRPQR (SEQ ID NO:

552)(A03.01)

SLWTTRPQRV (SEQ ID

NO: 553)(A02.01)

SPSAAAATL (SEQ ID NO:

554)(B07.02)

SPSAAAATLL (SEQ ID

NO: 555)(B07.02)

TLDALPAGCV (SEQ ID

NO: 556)(A02.01)

TVSTTTAPV (SEQ ID NO:

557)(A02.01)

VLSASILPA (SEQ ID NO:

558)(A02.01)

VLSASILPAA (SEQ ID NO:

559)(A02.01)

VPANCLFPA (SEQ ID NO:

560)(B07.02)

VPANCLFPAA (SEQ ID

NO: 561)(B07.02)

VPDPSCPTF (SEQ ID NO:

562)(B07.02)

VPGSIPLPA (SEQ ID NO:

563)(B07.02)

VPGSIPLPAV (SEQ ID NO:

564)(B07.02)

WVSGILSPL (SEQ ID NO:

565)(A02.01)

YTADSSLWTT (SEQ ID

NO: 566)(A02.01)

ARID1
T433fs

PCRAGRRVPWAASLIHSRF

APAGMVNRA (SEQ ID
STAD, UCEC,

A
A441fs

LLMDNKAPAGMVNRARLH

NO: 567)(B07.02)
BLCA, BRCA,

Y447fs

ITTSKVLTLSSSSHPTPSNHR

ASLHRRSYL (SEQ ID NO:
LUSC, CESC,

P483fs

PRPLMPNLRISSSHSLNHHS

568)(B08.01)
KIRC, UCS

P484fs

SSPLSLHTPSSHPSLHISSPR

ASLHRRSYLK (SEQ ID

P504fs

LHTPPSSRRHSSTPRASPPT

NO: 569)(A03.01)

S519fs

HSHRLSLLTSSSNLSSQHPR

FLLMDNKAPA (SEQ ID

H544fs

RSPSRLRILSPSLSSPSKLPIP

NO: 570)(A02.01)

P549fs

SSASLHRRSYLKIHLGLRHP

HPRRSPSRL (SEQ ID NO:

P554fs

QPPQ* (SEQ ID NO: 101)
571)(B07.02, B08.01)

Q563fs

HPSLHISSP (SEQ ID NO:

572)(B07.02)

HRRSYLKIHL (SEQ ID

NO: 573)(B08.01)

HSRFLLMDNK (SEQ ID

NO: 574)(A03.01)

KLPIPSSASL (SEQ ID NO:

575)(A02.01)

KVLTLSSSSH (SEQ ID NO:

576)(A03.01)

LIHSRFLLM (SEQ ID NO:

577)(B08.01)

LLMDNKAPA (SEQ ID

NO: 578)(A02.01)

LMDNKAPAGM (SEQ ID

NO: 579)(A02.01)

LPIPSSASL (SEQ ID NO:

580)(B07.02)

MPNLRISSS (SEQ ID NO:

581)(B07.02, B08.01)

MPNLRISSSH (SEQ ID NO:

582)(B07.02)

NLRISSSHSL (SEQ ID NO:

583)(B07.02, B08.01)

PPTHSHRLSL (SEQ ID NO:

584)(B07.02)

RAGRRVPWAA (SEQ ID

NO: 585)(B08.01)

RARLHITTSK (SEQ ID NO:

586)(A03.01)

RISSSHSLNH (SEQ ID NO:

587)(A03.01)

RLHTPPSSR (SEQ ID NO:

588)(A03.01)

RLHTPPSSRR (SEQ ID NO:

589)(A03.01)

RLRILSPSL (SEQ ID NO:

590)(A02.01, B07.02,

B08.01)

RPLMPNLRI (SEQ ID NO:

591)(B07.02)

RPRPLMPNL (SEQ ID NO:

592)(B07.02)

SASLHRRSYL (SEQ ID

NO: 593)(B07.02, B08.01)

SLHISSPRL (SEQ ID NO:

594)(A02.01)

SLHRRSYLK (SEQ ID NO:

595)(A03.01)

SLHRRSYLKI (SEQ ID NO:

596)(B08.01)

SLIHSRFLL (SEQ ID NO:

597)(A02.01)

SLIHSRFLLM (SEQ ID NO:

598)(A02.01, B08.01)

SLLTSSSNL (SEQ ID NO:

599)(A02.01)

SLNHHSSSPL (SEQ ID NO:

600)(A02.01, B07.02,

B08.01)

SLSSPSKLPI (SEQ ID NO:

601)(A02.01)

SPLSLHTPS (SEQ ID NO:

602)(B07.02)

SPLSLHTPSS (SEQ ID NO:

603)(B07.02)

SPPTHSHRL (SEQ ID NO:

604)(B07.02)

SPRLHTPPS (SEQ ID NO:

605)(B07.02)

SPRLHTPPSS (SEQ ID NO:

606)(B07.02)

SPSLSSPSKL (SEQ ID NO:

607)(B07.02)

SYLKIHLGL (SEQ ID NO:

608)(A24.02)

TPSNHRPRPL (SEQ ID NO:

609)(B07.02, B08.01)

TPSSHIPSLHI (SEQ ID NO:

610)(B07.02)

ARID1
A2137fs

RTNPTVRMRPHCVPFWTG

CVPFWTGRIL (SEQ ID
STAD, UCEC,

A
P2139fs

RILLPSAASVCPIPFEACHLC

NO: 611)(B07.02)
BLCA, BRCA,

L1970fs

QAMTLRCPNTQGCCSSWA

HCVPFWTGRIL (SEQ ID
LUSC, CESC,

V1994fs

S* (SEQ ID NO: 102)
NO: 612)(B07.02)
KIRC, UCS

ILLPSAASV (SEQ ID NO:

613)(A02.01)

ILLPSAASVC (SEQ ID NO:

614)(A02.01)

LLPSAASVCPI (SEQ ID

NO: 615)(A02.01)

LPSAASVCPI (SEQ ID NO:

616)(B07.02)

MRPHCVPF (SEQ ID NO:

617)(B08.01)

RILLPSAASV (SEQ ID NO:

618)(A02.01)

RMRPHCVPF (SEQ ID NO:

619)(A24.02, B07.02,

B08.01)

RMRPHCVPFW (SEQ ID

NO: 620)(A24.02)

RTNPTVRMR (SEQ ID NO:

621)(A03.01)

SVCPIPFEA (SEQ ID NO:

622)(A02.01)

TVRMRPHCV (SEQ ID NO:

623)(B08.01)

TVRMRPHCVPF (SEQ ID

NO: 624)(B08.01)

VPFWTGRIL (SEQ ID NO:

625)(B07.02)

VPFWTGRILL (SEQ ID

NO: 626)(B07.02)

VRMRPHCVPF (SEQ ID

NO: 627)(B08.01)

ARID1
N756fs

TNQALPKIEVICRGTPRCPS

AMVPRGVSM (SEQ ID
STAD, UCEC,

A
S764fs

TVPPSPAQPYLRVSLPEDRY

NO: 628)(B07.02, B08.01)
BLCA, BRCA,

T783fs

TQAWAPTSRTPWGAMVPR

AMVPRGVSMA (SEQ ID
LUSC, CESC,

Q799fs

GVSMAHKVATPGSQTIMPC

NO: 629)(A02.01)
KIRC, UCS

A817fs

PMPTTPVQAWLEA* (SEQ
AWAPTSRTPW (SEQ ID

ID NO: 103)
NO: 630)(A24.02)

CPMPTTPVQA (SEQ ID

NO: 631)(B07.02)

CPSTVPPSPA (SEQ ID NO:

632)(B07.02)

GAMVPRGVSM (SEQ ID

NO: 633)(B07.02, B08.01)

MPCPMPTTPV (SEQ ID

NO: 634)(B07.02)

MPTTPVQAW (SEQ ID

NO: 635)(B07.02)

MPTTPVQAWL (SEQ ID

NO: 636)(B07.02)

SLPEDRYTQA (SEQ ID

NO: 637)(A02.01)

SPAQPYLRV (SEQ ID NO:

638)(B07.02)

SPAQPYLRVS (SEQ ID

NO: 639)(B07.02)

TIMPCPMPT (SEQ ID NO:

640)(A02.01)

TPVQAWLEA (SEQ ID NO:

641)(B07.02)

TSRTPWGAM (SEQ ID

NO: 642)(B07.02)

VPPSPAQPYL (SEQ ID

NO: 643)(B07.02)

VPRGVSMAH (SEQ ID

NO: 644)(B07.02)

β2M
N62fs

RMERELKKWSIQTCLSART

CLSARTGLSI (SEQ ID NO:
CRC, STAD,

E67fs

GLSISCTTLNSPPLKKMSMP

645)(B08.01)
SKCM, HNSC

L74fs

AV* (SEQ ID NO: 104)
CTTLNSPPLK (SEQ ID NO:

F82fs

646)(A03.01)

T91fs

GLSISCTTL (SEQ ID NO:

E94fs

647)(A02.01)

SPPLKKMSM (SEQ ID NO:

648)(B07.02, B08.01)

TLNSPPLKK (SEQ ID NO:

649)(A03.01)

TTLNSPPLK (SEQ ID NO:

650)(A03.01)

TTLNSPPLKK (SEQ ID

NO: 651)(A03.01)

β2M
L13fs

LCSRYSLFLAWRLSSVLQR

LQRFRFTHV (SEQ ID NO:
CRC, STAD,

S14fs

FRFTHVIQQRMESQIS*

652)(B08.01)
SKCM, HNSC

(SEQ ID NO: 105)
LQRFRFTHVI (SEQ ID NO:

653)(B08.01)

RLSSVLQRF (SEQ ID NO:

654)(A24.02)

RLSSVLQRFR (SEQ ID

NO: 655)(A03.01)

VLQRFRFTHV (SEQ ID

NO: 656)(A02.01, B08.01)

CDH1
A691fs

RSACVTVKGPLASVGRHSL

ASVGRHSLSK (SEQ ID
ILC LumA

P708fs

SKQDCKFLPFWGFLEEFLL

NO: 657)(A03.01)
Breast Cancer

L711fs

C* (SEQ ID NO: 106)
KFLPFWGFL (SEQ ID NO:

658)(A24.02)

LASVGRHSL (SEQ ID NO:

659)(B07.02)

LPFWGFLEEF (SEQ ID

NO: 660)(B07.02)

PFWGFLEEF (SEQ ID NO:

661)(A24.02)

SVGRHSLSK (SEQ ID NO:

662)(A03.01)

CDH1
H121fs

IQWGTTTAPRPIRPPFLESK

APRPIRPPF (SEQ ID NO:
ILC LumA

P126fs

QNCSHFPTPLLASEDRRET

663)(B07.02)
Breast Cancer

H128fs

GLFLPSAAQKMKKAHFLK

APRPIRPPFL (SEQ ID NO:

N144fs

TWFRSNPTKTKKARFSTAS

664)(B07.02)

V157fs

LAKELTHPLLVSLLLKEKQ

AQKMKKAHFL (SEQ ID

P159fs

DG* (SEQ ID NO: 107)
NO: 665)(B08.01)

N166fs

FLPSAAQKM (SEQ ID NO:

N181fs

666)(A02.01)

F189fs

GLFLPSAAQK (SEQ ID

P201fs

NO: 667)(A03.01)

F205fs

HPLLVSLLL (SEQ ID NO:

668)(B07.02)

KAHFLKTWFR (SEQ ID

NO: 669)(A03.01)

KARFSTASL (SEQ ID NO:

670)(B07.02)

KMKKAHFLK (SEQ ID

NO: 671)(A03.01)

KTWFRSNPTK (SEQ ID

NO: 672)(A03.01)

LAKELTHPL (SEQ ID NO:

673)(B07.02, B08.01)

LAKELTHPLL (SEQ ID

NO: 674)(B08.01)

NPTKTKKARF (SEQ ID

NO: 675)(B07.02)

QKMKKAHFL (SEQ ID

NO: 676)(B08.01)

RFSTASLAK (SEQ ID NO:

677)(A03.01)

RPIRPPFLES (SEQ ID NO:

678)(B07.02)

RSNPTKTKK (SEQ ID NO:

679)(A03.01)

SLAKELTHPL (SEQ ID

NO: 680)(A02.01, B08.01)

TKKARFSTA (SEQ ID NO:

681)(B08.01)

CDH1
V114fs

PTDPFLGLRLGLHLQKVFH

GLRFWNPSR (SEQ ID NO:
ILC LumA

P127fs

QSHAEYSGAPPPPPAPSGLR

682)(A03.01)
Breast Cancer

V132fs

FWNPSRIAHISQLLSWPQKT

ISQLLSWPQK (SEQ ID

P160fs

EERLGYSSHQLPRK* (SEQ
NO: 683)(A03.01)

ID NO: 108)
RIAHISQLL (SEQ ID NO:

684)(A02.01)

RLGYSSHQL (SEQ ID NO:

685)(A02.01)

SQLLSWPQK (SEQ ID NO:

686)(A03.01)

SRIAHISQL (SEQ ID NO:

687)(B08.01)

WPQKTEERL (SEQ ID NO:

688)(B07.02)

YSSHQLPRK (SEQ ID NO:

689)(A03.01)

CDH1
L731fs

FCCSCCFFGGERWSKSPYC

CPQRMTPGTT (SEQ ID
ILC LumA

R749fs

PQRMTPGTTFITMMKKEAE

NO: 690)(B07.02)
Breast Cancer

E757fs

KRTRTLT* (SEQ ID NO:
EAEKRTRTL (SEQ ID NO:

G759fs
109)
691)(B08.01)

GTTFITMMK (SEQ ID NO:

692)(A03.01)

GTTFITMMKK (SEQ ID

NO: 693)(A03.01)

ITMMKKEAEK (SEQ ID

NO: 694)(A03.01)

RMTPGTTFI (SEQ ID NO:

695)(A02.01)

SPYCPQRMT (SEQ ID NO:

696)(B07.02)

TMMKKEAEK (SEQ ID

NO: 697)(A03.01)

TPGTTFITM (SEQ ID NO:

698)(B07.02)

TPGTTFITMM (SEQ ID

NO: 699)(B07.02)

TTFITMMKK (SEQ ID NO:

700)(A03.01)

CDH1
S19fs

WRRNCKAPVSLRKSVQTP

CPGATWREA (SEQ ID NO:
ILC LumA

E24fs

ARSSPARPDRTRRLPSLGVP

701)(B07.02)
Breast Cancer

S36fs

GQPWALGAAASRRCCCCC

CPGATWREAA (SEQ ID

RSPLGSARSRSPATLALTPR

NO: 702)(B07.02)

ATRSRCPGATWREAASWA

RSRCPGATWR (SEQ ID

E* (SEQ ID NO: 110)
NO: 703)(A03.01)

TPRATRSRC (SEQ ID NO:

704)(B07.02)

GATA3
P394fs

PGRPLQTHVLPEPHLALQP

HVLPEPHLAL (SEQ ID
Breast Cancer

P387fs

LQPHADHAHADAPAIQPVL

NO: 705)(B07.02)

S398fs

WTTPPLQHGHRHGLEPCS

RPLQTHVLPE (SEQ ID

H400fs

MLTGPPARVPAVPFDLHFC

NO: 706)(B07.02)

M401fs

RSSIMKPKRDGYMFLKAES

VLWTTPPLQH (SEQ ID

S408fs

KIMFATLQRSSLWCLCSNH*

NO: 707)(A03.01)

P409fs
(SEQ ID NO: 111)

S408fs

P409fs

T419fs

H424fs

P425fs

S427fs

F431fs

S430fs

H434fs

H435fs

S438fs

M443fs

G444fs

*445fs

GATA3
P426fs

PRPRRCTRHPACPLDHTTPP

APSESPCSPF (SEQ ID NO:
Breast Cancer

H434fs

AWSPPWVRALLDAHRAPS

708)(B07.02)

P433fs

ESPCSPFRLAFLQEQYHEA*

CPLDHTTPPA (SEQ ID

T441fs
(SEQ ID NO: 112)
NO: 709)(B07.02)

FLQEQYHEA (SEQ ID NO:

710)(A02.01, B08.01)

RLAFLQEQYH (SEQ ID

NO: 711)(A03.01)

SPCSPFRLAF (SEQ ID NO:

712)(B07.02)

SPPWVRALL (SEQ ID NO:

713)(B07.02)

YPACPLDHTT (SEQ ID

NO: 714)(B07.02)

MLL2
P519fs

TRRCHCCPHLRSHPCPHHL

ALHLRSCPC (SEQ ID NO:
STAD, BLCA,

E524fs

RNHPRPHHLRHHACHHHL

715)(B08.01)
CRC, HNSC,

P647fs

RNCPHPHFLRHCTCPGRWR

CLHHRRHLV (SEQ ID NO:
BRCA

S654fs

NRPSLRRLRSLLCLPHLNH

716)(B08.01)

L656fs

HLFLHWRSRPCLHRKSHPH

CLHHRRHLVC (SEQ ID

R755fs

LLHLRRLYPHHLKHRPCPH

NO: 717)(B08.01)

L761fs

HLKNLLCPRHLRNCPLPRH

CLHRKSHPHL (SEQ ID

Q773fs

LKHLACLHHLRSHPCPLHL

NO: 718)(B08.01)

KSHPCLHHRRHLVCSHHLK

CLRSHACPP (SEQ ID NO:

SLLCPLHLRSLPFPHHLRHH

719)(B08.01)

ACPHHLRTRLCPHHLKNHL

CLRSHTCPP (SEQ ID NO:

CPPHLRYRAYPPCLWCHAC

720)(B08.01)

LHRLRNLPCPHRLRSLPRPL

CLWCHACLH (SEQ ID

HLRLHASPHHLRTPPHPHH

NO. 721)(A03.01)

LRTHLLPHHRRTRSCPCRW

CPHHLKNHL (SEQ ID NO:

RSHPCCHYLRSRNSAPGPR

722)(B07.02)

GRTCHPGLRSRTCPPGLRS

CPHHLKNLL (SEQ ID NO:

HTYLRRLRSHTCPPSLRSH

723)(B07.02)

AYALCLRSHTCPPRLRDHI

CPHHLRTRL (SEQ ID NO:

CPLSLRNCTCPPRLRSRTCL

724)(B07.02, B08.01)

LCLRSHACPPNLRNHTCPPS

CPLHLRSLPF (SEQ ID NO:

LRSHACPPGLRNRICPLSLR

725)(B07.02, B08.01)

SHPCPLGLKSPLRSQANAL

CPLPRHLKHL (SEQ ID

HLRSCPCSLPLGNHPYLPCL

NO. 726)(B07.02, B08.01)

ESQPCLSLGNHLCPLCPRSC

CPLSLRSHPC (SEQ ID NO:

RCPHLGSHPCRLS* (SEQ ID
727)(B07.02)

NO: 113)
CPRHLRNCPL (SEQ ID

NO. 728)(B07.02, B08.01)

FPHHLRHHA (SEQ ID NO:

729)(B07.02, B08.01)

FPHHLRHHAC (SEQ ID

NO: 730)(B07.02, B08.01)

GLRSRTCPP (SEQ ID NO:

731)(B08.01)

HACLHRLRNL (SEQ ID

NO: 732)(B08.01)

HLACLHHLR (SEQ ID NO:

733)(A03.01)

HLCPPHLRY (SEQ ID NO:

734)(A03.01)

HLCPPHLRYR (SEQ ID

NO: 735)(A03.01)

HLKHLACLH (SEQ ID NO:

736)(A03.01)

HLKHRPCPH (SEQ ID NO:

737)(B08.01)

HLKNHLCPP (SEQ ID NO:

738)(B08.01)

HLKSHPCLH (SEQ ID NO:

739)(A03.01)

HLKSLLCPL (SEQ ID NO:

740)(A02.01, B08.01)

HLLHLRRLY (SEQ ID NO:

741)(A03.01)

HLRNCPLPR (SEQ ID NO:

742)(A03.01)

HLRNCPLPRH (SEQ ID

NO: 743)(A03.01)

HLRRLYPHHL (SEQ ID

NO: 744)(B08.01)

HLRSHPCPL (SEQ ID NO:

745)(B07.02, B08.01)

HLRSHPCPLH (SEQ ID

NO: 746)(A03.01)

HLRSLPFPH (SEQ ID NO:

747)(A03.01)

HLRTRLCPH (SEQ ID NO:

748)(A03.01, B08.01)

HLVCSHHLK (SEQ ID NO:

749)(A03.01)

HPCLHHRRHL (SEQ ID

NO: 750)(B07.02, B08.01)

HPGLRSRTC (SEQ ID NO:

751)(B07.02)

HPHLLHLRRL (SEQ ID

NO: 752)(B07.02, B08.01)

HRKSHPHLL (SEQ ID NO:

753)(B08.01)

HRRTRSCPC (SEQ ID NO:

754)(B08.01)

KSHPHLLHLR (SEQ ID

NO: 755)(A03.01)

KSLLCPLHLR (SEQ ID

NO: 756)(A03.01)

LLCPLHLRSL (SEQ ID NO:

757)(A02.01, B08.01)

LLHLRRLYPH (SEQ ID

NO: 758)(B08.01)

LPRHLKHLA (SEQ ID NO:

759)(B07.02)

LPRHLKHLAC (SEQ ID

NO: 760)(B07.02, B08.01)

LRRLRSHTC (SEQ ID NO:

761)(B08.01)

LRRLYPHHL (SEQ ID NO:

762)(B08.01)

LVCSHHLKSL (SEQ ID

NO: 763)(B08.01)

NLRNHTCPPS (SEQ ID

NO: 764)(B08.01)

PLHLRSLPF (SEQ ID NO:

765)(B08.01)

RLCPHHLKNH (SEQ ID

NO: 766)(A03.01)

RLYPHHLKH (SEQ ID NO:

767)(A03.01)

RLYPHHLKHR (SEQ ID

NO: 768)(A03.01)

RPCPHHLKNL (SEQ ID

NO: 769)(B07.02)

RSHPCPLHLK (SEQ ID

NO: 770)(A03.01)

RSLPFPHHLR (SEQ ID NO:

771)(A03.01)

RTRLCPHHL (SEQ ID NO:

772)(B07.02)

RTRLCPHHLK (SEQ ID

NO: 773)(A03.01)

SLLCPLHLR (SEQ ID NO:

774)(A03.01)

SLRSHACPP (SEQ ID NO:

775)(B08.01)

SPLRSQANA (SEQ ID NO:

776)(B07.02)

YLRRLRSHT (SEQ ID NO:

777)(B08.01)

YPHHLKHRPC (SEQ ID

NO: 778)(B07.02, B08.01)

PTEN
I122fs

SWKGTNWCNDMCIFITSGQ

FITSGQIFK (SEQ ID NO:
UCEC, PRAD,

I135fs

IFKGTRGPRFLWGSKDQRQ

779)(A03.01)
SKCM, STAD,

A148fs

KGSNYSQSEALCVLL*

IFITSGQIF (SEQ ID NO:
BRCA, LUSC,

L152fs
(SEQ ID NO: 114)
780)(A24.02)
KIRC, LIHC,

D162fs

SQSEALCVL (SEQ ID NO:
KIRP, GBM

I168fs

781)(A02.01)

SQSEALCVLL (SEQ ID

NO: 782)(A02.01)

PTEN
L265fs

KRTKCFTFG* (SEQ ID NO:

UCEC, PRAD,

K266fs
115)

SKCM, STAD,

BRCA, LUSC,

KIRC, LIHC,

KIRP, GBM

PTEN
A39fs

PIFIQTLLLWDFLQKDLKAY

AYTGTILMM (SEQ ID NO:
UCEC, PRAD,

E40fs

TGTILMM* (SEQ ID NO:
783)(A24.02)
SKCM, STAD,

V45fs
116)
DLKAYTGTIL (SEQ ID
BRCA, LUSC,

R47fs

NO: 784)(B08.01)
KIRC, LIHC,

N48fs

KIRP, GBM

PTEN
T319fs

QKMILTKQIKTKPTDTFLQI

ILTKQIKTK (SEQ ID NO:
UCEC, PRAD,

T321fs

LR* (SEQ ID NO: 117)
785)(A03.01)
SKCM, STAD,

K327fs

KMILTKQIK (SEQ ID NO:
BRCA, LUSC,

A328fs

786)(A03.01)
KIRC, LIHC,

A333fs

KPTDTFLQI (SEQ ID NO:
KIRP, GBM

787)(B07.02)

KPTDTFLQIL (SEQ ID NO:

788)(B07.02)

MILTKQIKTK (SEQ ID

NO: 789)(A03.01)

PTEN
N63fs

GFWIQSIKTITRYTIFVLKDI

ITRYTIFVLK (SEQ ID NO:
UCEC, PRAD,

E73fs

MTPPNLIAELHNILLKTITH

790)(A03.01)
SKCM, STAD,

A86fs

HS* (SEQ ID NO: 118)
LIAELHNIL (SEQ ID NO:
BRCA, LUSC,

N94fs

791)(A02.01)
KIRC, LIHC,

LIAELHNILL (SEQ ID NO:
KIRP, GBM

792)(A02.01)

MTPPNLIAEL (SEQ ID NO:

793)(A02.01)

NLIAELHNI (SEQ ID NO:

794)(A02.01)

NLIAELHNIL (SEQ ID NO:

795)(A02.01)

RYTIFVLKDI (SEQ ID NO:

796)(A24.02)

TITRYTIFVL (SEQ ID NO:

797)(A02.01)

TPPNLIAEL (SEQ ID NO:

798)(B07.02)

PTEN
T202fs

NYSNVQWRNLQSSVCGLP

FLQFRTHTT (SEQ ID NO:
UCEC, PRAD,

G209fs

AKGEDIFLQFRTHTTGRQV

799)(A02.01, B08.01)
SKCM, STAD,

C211fs

HVL* (SEQ ID NO: 119)
LPAKGEDIFL (SEQ ID NO:
BRCA, LUSC,

I224fs

800)(B07.02)
KIRC, LIHC,

G230fs

LQFRTHTTGR (SEQ ID
KIRP, GBM

P231fs

NO: 801)(A03.01)

R233fs

NLQSSVCGL (SEQ ID NO:

D236fs

802)(A02.01)

SSVCGLPAK (SEQ ID NO:

803)(A03.01)

VQWRNLQSSV (SEQ ID

NO: 804)(A02.01)

PTEN
G251fs

YQSRVLPQTEQDAKKGQN

GQNVSLLGK (SEQ ID NO:
UCEC, PRAD,

E256fs

VSLLGKYILHTRTRGNLRK

805)(A03.01)
SKCM, STAD,

K260fs

SRKWKSM* (SEQ ID NO:
HTRTRGNLRK (SEQ ID
BRCA, LUSC,

Q261fs
120)
NO: 806)(A03.01)
KIRC, LIHC,

L265fs

ILHTRTRGNL (SEQ ID
KIRP, GBM

M270fs

NO: 807)(B08.01)

H272fs

KGQNVSLLGK (SEQ ID

T286fs

NO: 808)(A03.01)

E288fs

LLGKYILHT (SEQ ID NO:

809)(A02.01)

LRKSRKWKSM (SEQ ID

NO: 810)(B08.01)

SLLGKYILH (SEQ ID NO:

811)(A03.01)

SLLGKYILHT (SEQ ID NO:

812)(A02.01)

TP53
A70fs

SSQNARGCSPRGPCTSSSYT

CTSPLLAPV (SEQ ID NO:
BRCA, CRC,

P72fs

GGPCTSPLLAPVIFCPFPEN

813)(A02.01)
LUAD, PRAD,

A76fs

LPGQLRFPSGLLAFWDSQV

FPENLPGQL (SEQ ID NO:
HNSC, LUSC,

A79fs

CDLHVLPCPQQDVLPTGQD

814)(B07.02)
PAAD, STAD,

P89fs

LPCAAVG* (SEQ ID NO:
GLLAFWDSQV (SEQ ID
BLCA, OV,

W91fs
121)
NO: 815)(A02.01)
LIHC, SKCM,

S96fs

IFCPFPENL (SEQ ID NO:
UCEC, LAML,

V97fs

816)(A24.02)
UCS, KICH,

V97fs

LLAFWDSQV (SEQ ID NO:
GBM, ACC

G108fs

817)(A02.01)

G117fs

LLAPVIFCP (SEQ ID NO:

S121fs

818)(A02.01)

V122fs

LLAPVIFCPF (SEQ ID NO:

C124fs

819)(A02.01, A24.02)

K139fs

LPCPQQDVL (SEQ ID NO:

V143fs

820)(B07.02)

RFPSGLLAF (SEQ ID NO:

821)(A24.02)

RFPSGLLAFW (SEQ ID

NO: 822)(A24.02)

SPLLAPVIF (SEQ ID NO:

823)(B07.02)

SPRGPCTSS (SEQ ID NO:

824)(B07.02)

SPRGPCTSSS (SEQ ID NO:

825)(B07.02)

SQVCDLHVL (SEQ ID NO:

826)(A02.01)

VIFCPFPENL (SEQ ID NO:

827)(A02.01)

TP53
V173fs

GAAPTMSAAQIAMVWPLL

AMVWPLLSI (SEQ ID NO:
BRCA, CRC,

H178fs

SILSEWKEICVWSIWMTET

828)(A02.01)
LUAD, PRAD,

D186fs

LFDIVWWCPMSRLRLALTV

AMVWPLLSIL (SEQ ID
HNSC, LUSC,

H193fs

PPSTTTTCVTVPAWAA*

NO: 829)(A02.01)
PAAD, STAD,

L194fs
(SEQ ID NO: 122)
AQIAMVWPL (SEQ ID NO:
BLCA, OV,

E198fs

830)(A02.01, A24.02)
LIHC, SKCM,

V203fs

AQIAMVWPLL (SEQ ID
UCEC, LAML,

E204fs

NO: 831)(A02.01)
UCS, KICH,

L206fs

CPMSRLRLA (SEQ ID NO:
GBM, ACC

D207fs

832)(B07.02, B08.01)

N210fs

CPMSRLRLAL (SEQ ID

T211fs

NO: 833)(B07.02, B08.01)

F212fs

IAMVWPLLSI (SEQ ID

V225fs

NO: 834)(A02.01, A24.02,

S241fs

B08.01)

ILSEWKEICV (SEQ ID NO:

835)(A02.01)

IVWWCPMSR (SEQ ID

NO: 836)(A03.01)

IVWWCPMSRL (SEQ ID

NO: 837)(A02.01)

IWMTETLFDI (SEQ ID NO:

838)(A24.02)

LLSILSEWK (SEQ ID NO:

839)(A03.01)

MSAAQIAMV (SEQ ID

NO: 840)(A02.01)

MSRLRLALT (SEQ ID NO:

841)(B08.01)

MSRLRLALTV (SEQ ID

NO: 842)(B08.01)

MVWPLLSIL (SEQ ID NO:

843)(A02.01)

RLALTVPPST (SEQ ID NO:

844)(A02.01)

TLFDIVWWC (SEQ ID NO:

845)(A02.01)

TLFDIVWWCP (SEQ ID

NO: 846)(A02.01)

TMSAAQIAMV (SEQ ID

NO: 847)(A02.01)

VWSIWMTETL (SEQ ID

NO: 848)(A24.02)

WMTETLFDI (SEQ ID NO:

849)(A02.01, A24.02)

WMTETLFDIV (SEQ ID

NO: 850)(A01.01, A02.01)

TP53
R248fs

TGGPSSPSSHWKTPVVIYW

ALRCVFVPV (SEQ ID NO:
BRCA, CRC,

P250fs

DGTALRCVFVPVLGETGAQ

851)(A02.01, B08.01)
LUAD, PRAD,

S260fs

RKRISARKGSLTTSCPQGAL

ALRCVFVPVL (SEQ ID
HNSC, LUSC,

N263fs

SEHCPTTPAPLPSQRRNHW

NO: 852)(A02.01, B08.01)
PAAD, STAD,

G266fs

MENISPFRSVGVSASRCSES*

ALSEHCPTT (SEQ ID NO:
BLCA, OV,

N268fs
(SEQ ID NO: 123)
853)(A02.01)
LIHC, SKCM,

V272fs

AQRKRISARK (SEQ ID
UCEC, LAML,

V274fs

NO: 854)(A03.01)
UCS, KICH,

P278fs

GAQRKRISA (SEQ ID NO:
GBM, ACC

D281fs

855)(B08.01)

R282fs

HWMENISPF (SEQ ID NO:

T284fs

856)(A24.02)

E285fs

LPSQRRNHW (SEQ ID NO:

L289fs

857)(B07.02)

K292fs

LPSQRRNHWM (SEQ ID

P301fs

NO: 858)(B07.02, B08.01)

S303fs

NISPFRSVGV (SEQ ID NO:

T312fs

859)(A02.01)

S314fs

RISARKGSL (SEQ ID NO:

K319fs

860)(B07.02, B08.01)

K320fs

SPFRSVGVSA (SEQ ID

P322fs

NO: 861)(B07.02)

Y327fs

SPSSHWKTPV (SEQ ID

F328fs

NO: 862)(B07.02, B08.01)

L330fs

TALRCVFVPV (SEQ ID

R333fs

NO: 863)(A02.01)

R335fs

VIYWDGTAL (SEQ ID NO:

R337fs

864)(A02.01)

E339fs

VIYWDGTALR (SEQ ID

NO: 865)(A03.01)

VLGETGAQRK (SEQ ID

NO: 866)(A03.01)

TP53
S149fs

FHTPARHPRPRHGHLQAVT

HPRPRHGHL (SEQ ID NO:
BRCA, CRC,

P151fs

AHDGGCEALPPP* (SEQ ID
867)(B07.02, B08.01)
LUAD, PRAD,

P152fs
NO: 124)
HPRPRHGHLQ (SEQ ID
HNSC, LUSC,

V157fs

NO: 868)(B07.02)
PAAD, STAD,

Q165fs

RPRHGHLQA (SEQ ID NO:
BLCA, OV,

S166fs

869)(B07.02)
LIHC, SKCM,

H168fs

RPRHGHLQAV (SEQ ID
UCEC, LAML,

V173fs

NO: 870)(B07.02, B08.01)
UCS, KICH,

GBM, ACC

TP53
P47fs

CCPRTILNNGSLKTQVQMK

GSLKTQVQMK (SEQ ID
BRCA, CRC,

D48fs

LPECQRLLPPWPLHQQLLH

NO: 871)(A03.01)
LUAD, PRAD,

D49fs

RRPLHQPPPGPCHLLSLPRK

PPGPCHLLSL (SEQ ID NO:
HNSC, LUSC,

Q52fs

PTRAATVSVWASCILGQPS

872)(B07.02)
PAAD, STAD,

F54fs

L* (SEQ ID NO: 125)
RTILNNGSLK (SEQ ID
BLCA, OV,

E56fs

NO: 873)(A03.01)
LIHC, SKCM,

P58fs

SLKTQVQMK (SEQ ID
UCEC, LAML,

P60fs

NO: 874)(A03.01)
UCS, KICH,

E62fs

SLKTQVQMKL (SEQ ID
GBM, ACC

M66fs

NO: 875)(B08.01)

P72fs

TILNNGSLK (SEQ ID NO:

V73fs

876)(A03.01)

P75fs

A78fs

P82fs

P85fs

S96fs

P98fs

T102fs

Y103fs

G108fs

F109fs

R110fs

G117fs

TP53
L26fs

VRKHFQTYGNYFLKTTFCP

CPPCRPKQWM (SEQ ID
BRCA, CRC,

P27fs

PCRPKQWMI* (SEQ ID NO:
NO: 877)(B07.02)
LUAD, PRAD,

P34fs
126)
TTFCPPCRPK (SEQ ID NO:
HNSC, LUSC,

P36fs

878)(A03.01)
PAAD, STAD,

A39fs

BLCA, OV,

Q38fs

LIHC, SKCM,

UCEC, LAML,

UCS, KICH,

GBM, ACC

TP53
C124fs

LARTPLPSTRCFANWPRPA

CFANWFPRPAL (SEQ ID
BRCA, CRC,

L130fs

LCSCGLIPHPRPAPASAPWP

NO: 879)(A24.02)
LUAD, PRAD,

N131fs

STSSHST* (SEQ ID NO: 127)
FANWFPRPAL (SEQ ID NO:
HNSC, LUSC,

C135fs

880)(B07.02, B08.01)
PAAD, STAD,

K139fs

GLIPHPRPA (SEQ ID NO:
BLCA, OV,

A138fs

881)(A02.01)
LIHC, SKCM,

T140fs

HPRPAPASA (SEQ ID NO:
UCEC, LAML,

V143fs

882)(B07.02, B08.01)
UCS, KICH,

Q144fs

HPRPAPASAP (SEQ ID
GBM, ACC

V147fs

NO: 883)(B07.02)

T150fs

IPHPRPAPA (SEQ ID NO:

P151fs

884)(B07.02, B08.01)

P152fs

IPHPRPAPAS (SEQ ID NO:

G154fs

885)(B07.02)

R156fs

RPALCSCGL (SEQ ID NO:

R158fs

886)(B07.02)

A161fs

RPALCSCGLI (SEQ ID NO:

887)(B07.02)

TPLPSTRCF (SEQ ID NO:

888)(B07.02)

WPRPALCSC (SEQ ID NO:

889)(B07.02)

WPRPALCSCG (SEQ ID

NO: 890)(B07.02)

VHL
L178fs

ELQETGHRQVALRRSGRPP

ALRRSGRPPK (SEQ ID
KIRC, KIRP

D179fs

KCAERPGAADTGAHCTST

NO: 891)(A03.01)

L184fs

DGRLKISVETYTVSSQLLM

GLVPSLVSK (SEQ ID NO:

T202fs

VLMSLDLDTGLVPSLVSKC

892)(A03.01)

R205fs

LILRVK* (SEQ ID NO: 128)
KISVETYTV (SEQ ID NO:

D213fs

893)(A02.01)

G212fs

LLMVLMSLDL (SEQ ID

NO: 894)(A02.01, B08.01)

LMSLDLDTGL (SEQ ID

NO: 895)(A02.01)

LMVLMSLDL (SEQ ID NO:

896)(A02.01)

LVSKCLILRV (SEQ ID NO:

897)(A02.01)

QLLMVLMSL (SEQ ID NO:

898)(A02.01, B08.01)

RPGAADTGA (SEQ ID NO:

899)(B07.02)

RPGAADTGAH (SEQ ID

NO: 900)(B07.02)

SLDLDTGLV (SEQ ID NO:

901)(A02.01)

SLVSKCLIL (SEQ ID NO:

902)(A02.01, B08.01)

SQLLMVLMSL (SEQ ID

NO: 903)(A02.01)

TVSSQLLMV (SEQ ID NO:

904)(A02.01)

TYTVSSQLL (SEQ ID NO:

905)(A24.02)

TYTVSSQLLM (SEQ ID

NO: 906)(A24.02)

VLMSLDLDT (SEQ ID NO:

907)(A02.01)

VPSLVSKCL (SEQ ID NO:

908)(B07.02)

VSKCLILRVK (SEQ ID

NO: 909)(A03.01)

YTVSSQLLM (SEQ ID NO:

910)(A01.01)

YTVSSQLLMV (SEQ ID

NO: 911)(A02.01)

VHL
L158fs

KSDASRLSGA* (SEQ ID

KIRC, KIRP

K159fs
NO: 129)

R161fs

Q164fs

VHL
P146fs

RTAYFCQYHTASVYSERA

FCQYHTASV (SEQ ID NO:
KIRC, KIRP

I147fs

MPPGCPEPSQA* (SEQ ID
912)(B08.01)

F148fs
NO: 130)

L158fs

VHL
S68fs

TRASPPRSSSAIAVRASCCP

CPYGSTSTA (SEQ ID NO:
KIRC, KIRP

S72fs

YGSTSTASRSPTQRCRLAR

913)(B07.02)

I75fs

AAASTATEVTFGSSEMQGH

CPYGSTSTAS (SEQ ID

S80fs

TMGFWLTKLNYLCHLSML

NO: 914)(B07.02)

P86fs

TDSLFLPISHCQCIL* (SEQ
LARAAASTAT (SEQ ID

P97fs
ID NO: 131)
NO: 915)(B07.02)

I109fs

MLTDSLFLP (SEQ ID NO:

H115fs

916)(A02.01)

L116fs

PPRSSSAIAV (SEQ ID NO:

G123fs

917)(B07.02)

T124fs

RAAASTATEV (SEQ ID

N131fs

NO: 918)(B07.02)

L135fs

SPPRSSSAI (SEQ ID NO:

V137fs

919)(B07.02)

G144fs

SPPRSSSAIA (SEQ ID NO:

D143fs

920)(B07.02)

I147fs

SPTQRCRLA (SEQ ID NO:

921)(B07.02)

TQRCRLARA (SEQ ID NO:

922)(B08.01)

TQRCRLARAA (SEQ ID

NO: 923)(B08.01)

VHL
K171fs

SSLRITGDWTSSGRSTKIWK

KIWKTTQMCR (SEQ ID
KIRC, KIRP

P172fs

TTQMCRKTWSG* (SEQ ID
NO: 924)(A03.01)

N174fs
NO: 132)
WTSSGRSTK (SEQ ID NO:

L178fs

925)(A03.01)

D179fs

L188fs

VHL
V62fs

RRRRGGVGRRGVRPGRVR

ALGELARAL (SEQ ID NO:
KIRC, KIRP

V66fs

PGGTGRRGGDGGRAAAAR

926)(A02.01)

Q73fs

AALGELARALPGHLLQSQS

AQLRRRAAA (SEQ ID NO:

V84fs

ARRAARMAQLRRRAAALP

927)(B08.01)

F91fs

NAAAWHGPPHPQLPRSPLA

AQLRRRAAAL (SEQ ID

T100fs

LQRCRDTRWASG* (SEQ ID
NO: 928)(B08.01)

P103fs
NO: 133)
ARRAARMAQL (SEQ ID

S111fs

NO: 929)(B08.01)

L116fs

HPQLPRSPL (SEQ ID NO:

H115fs

930)(B07.02, B08.01)

D126fs

HPQLPRSPLA (SEQ ID

NO. 931)(B07.02)

LARALPGHL (SEQ ID NO:

932)(B07.02)

LARALPGHLL (SEQ ID

NO: 933)(B07.02)

MAQLRRRAA (SEQ ID

NO: 934)(B07.02, B08.01)

MAQLRRRAAA (SEQ ID

NO: 935)(B07.02, B08.01)

QLRRRAAAL (SEQ ID NO:

936)(B07.02, B08.01)

RAAALPNAAA (SEQ ID

NO: 937)(B07.02)

RMAQLRRRAA (SEQ ID

NO: 938)(B07.02, B08.01)

SQSARRAARM (SEQ ID

NO: 939)(B08.01)

TABLE 1D

CRYPTIC EXON

AR-v7
cryptic
SCKVFFKRAAEGKQKYLC
GMTLGEKFRV (SEQ ID
Prostate Cancer,

final
ASRNDCTIDKFRRKNCPSC
NO: 940) (A02:01)
Castration-

exon
RLRKCYEAGMTLGEKFRV
RVGNCKEILK (SEQ ID
resistant

GNCKEILKMTRP* (SEQ ID
NO: 941) (A03.01)
Prostate Cancer

NO: 134)

TABLE 1E

OUT OF FRAME FUSIONS

AC011997.1:
AC011997.1:
MAGAPPPASLPPCSLISDCC
GPSEPGNNI (SEQ ID NO:
LUSC, Breast

LRRC69
LRRC69
ASNQRDSVGVGPSEP:G: custom-character

942) (B07.02)
Cancer, Head

*out-of-

custom-character

(SEQ ID
KICNESASRK (SEQ ID
and Neck

frame
NO: 135)
NO: 943) (A03.01)
Cancer, LUAD

EEF1DP3
EEF1DP3:
HGWRPFLPVRARSRWNRR
GIQVLNVSLK (SEQ ID
Breast Cancer

FRY
LDVTVANGR:S: custom-character

NO: 944) (A03.01)

*out-of-

custom-character

IQVLNVSLK (SEQ ID NO:

custom-character

945) (A03.01)

(SEQ ID NO: 136)
KSSSNVISY (SEQ ID NO:

946) (A01.01, A03.01)

KYGWSLLRV (SEQ ID

NO: 947) (A24.02)

RSWKYGWSL (SEQ ID

NO: 948) (A02.01)

SLKSSSNVI (SEQ ID NO:

949) (B08.01)

SWKYGWSLL (SEQ ID

NO: 950) (A24.02)

TVANGRSWK (SEQ ID

NO: 951) (A03.01)

VPQVNGIQV (SEQ ID NO:

952) (B07.02)

VPQVNGIQVL (SEQ ID

NO: 953) (B07.02)

VTVANGRSWK (SEQ ID

NO: 954) (A03.01)

WSLLRVPQV (SEQ ID NO:

955) (B08.01)

MAD1L1:
MAD1L1:
RLKEVFQTKIQEFRKACYT
HPGDCLIFKL (SEQ ID NO:
CLL

MAFK
MAFK
LTGYQIDITTENQYRLTSLY
956) (B07.02)

AEHPGDCLIFK:: custom-character

KLRVPGSSV (SEQ ID NO:

custom-character

(SEQ ID NO:
957) (B07.02)

137)
KLRVPGSSVL (SEQ ID

NO: 958) (B07.02)

RVPGSSVLV (SEQ ID NO:

959) (A02.01)

SVLVTVPGL (SEQ ID NO:

960) (A02.01)

VPGSSVLVTV (SEQ ID

NO: 961) (B07.02)

PPP1R1B:
PPP1R1B:
AEVLKVIRQSAGQKTTCGQ
ALLLRPRPPR (SEQ ID NO:
Breast Cancer

STARD3
STARD3
GLEGPWERPPPLDESERDG
962) (A03.01)

GSEDQVEDPALS:A: custom-character

ALSALLLRPR (SEQ ID

custom-character

NO: 963) (A03.01)

custom-character

(SEQ ID NO: 138)

TABLE 1F

IN-FRAME DELETIONS and FUSIONS

BCR:ABL
BCR:ABL
ERAEWRENIREQQKKCFRS
LTINKEEAL (SEQ ID NO:
CML, AML

FSLTSVELQMLTNSCVKLQ
964) (A02.01, B08.01)

TVHSIPLTINKE::EALQRPV

ASDFEPQGLSEAARWNSK

ENLLAGPSENDPNLFVAL

YDFVASG (SEQ ID NO: 139)

BCR:ABL
BCR:ABL
ELQMLTNSCVKLQTVHSIP
IVHSATGFK (SEQ ID NO:
CML, AML

LTINKEDDESPGLYGFLNVI
965) (A03.01)

VHSATGFKQSS:K:ALQRPV
ATGFKQSSK (SEQ ID NO:

ASDFEPQGLSEAARWNSK

966) (A03.01)

ENLLAGPSENDPNLFVAL

YDFVASGD (SEQ ID NO:

140)

C11orf95:RELA
C11orf95:RELA
ISNSWDAHLGLGACGEAEG
ELFPLIFPA (SEQ ID NO:
Supretentorial

LGVQGAEEEEEEEEEEEEE
967) (A02.01, B08.01)
ependyomas

GAGVPACPPKGP:E:LFPLIF
KGPELFPLI (SEQ ID NO:

PAEPAQASGPYVEIIEQPK

968) (A02.01, A24.02)

QRGMRFRYKCEGRSAGSI

KGPELFPLIF (SEQ ID NO:

PGERSTD (SEQ ID NO: 141)
969) (A24.02)

CBFB:MYH11
(variant
LQRLDGMGCLEFDEERAQ

AML

“type a”)
QEDALAQQAFEEARRRTRE

FEDRDRSHREEME::VHELE

KSKRALETQMEEMKTQL

EELEDELQATEDAKLRLE

VNMQALKGQF (SEQ ID

NO: 142)

CD74:ROS1
(exon6:exon32)
KGSFPENLRHLKNTMETID
KPTDAPPKAGV (SEQ ID
NSCLC,

WKVFESWMEIHWILFEMS
NO: 970) (B07.02)
Crizotinib

RHSLEQKPTDAPPK::AGVP

resistance

NKPGIPKLLEGSKNSIQW

EKAEDNGCRITYYILEIRK

STSNNLQNQ (SEQ ID NO:

143)

EGFR
EGFRvIII
MRPSGTAGAALLALLAAL
ALEEKKGNYV (SEQ ID
GBM

(internal
CPASRALEEKK:G:NYVVTD
NO: 971) (A02.01)

deletion)
HGSCVRACGADSYEMEED

GVRKCKKCEGPCRKVCNGI

GIGEFKD (SEQ ID NO: 144)

EGFR:SEPT14
EGFR:SEPT14
LPQPPICTIDVYMIMVKCW
IQLQDKFEHL (SEQ ID
GBM, Glioma,

MIDADSRPKFRELIIEFSKM
NO: 972) (A02.01, B08.01)
Head and Neck

ARDPQRYLVIQ::LQDKFEH
QLQDKFEHL (SEQ ID NO:
Cancer

LKMQQEEIRKLEEEKKQ

973) (A02.01, B08.01)

LEGEHDFYKMKAASEAL

QLQDKFEHLK (SEQ ID

QTQLSTD (SEQ ID NO: 145)
NO: 974) (A03.01)

YLVIQLQDKF (SEQ ID

NO: 975) (A02.01, A24.02)

EML4:ALK
EML4:ALK
SWENSDDSRNKLSKIPSTPK
QVYRRKHQEL (SEQ ID
NSCLC

LIPKVTKTADKHKDVIINQ
NO: 976) (B08.01)

AKMSTREKNSQ:V:YRRKH
STREKNSQV (SEQ ID NO:

QELQAMQMELQSPEYKL

977) (B08.01)

SKLRTSTIMTDYNPNYCF

VYRRKHQEL (SEQ ID NO:

AGKTSSISDL (SEQ ID NO:
978) (A24.02, B08.01)

146)

FGFR3:TACC3
FGFR3:TACC3
EGHRMDKPANCTHDLYMI
VLTVTSTDV (SEQ ID NO:
Bladder Cancer,

MRECWHAAPSQRPTFKQL
979) (A02.01)
LUSC

VEDLDRVLTVTSTD::VKAT
VLTVTSTDVK (SEQ ID

QEENRELRSRCEELHGKN

NO: 980) (A03.01)

LELGKIMDRFEEVVYQA

MEEVQKQKELS (SEQ ID

NO: 147)

NAB:STAT6
NAB:STAT6 “”
RDNTLLLRRVELFSLSRQV
IMSLWGLVS (SEQ ID NO:
Solitary fibrous

ARESTYLSSLKGSRLEIPEEL
981) (A02.01)
tumors

GGPPLKKLKQE::ATSKSQI
IMSLWGLVSK (SEQ ID

MSLWGLVSKMPPEKVQR

NO: 982) (A03.01)

LYVDFPQHLRHLLGDWL

KLKQEATSK (SEQ ID NO:

ESQPWEFLVGSDAFCC

983) (A03.01)

(SEQ ID NO: 148)
QIMSLWGLV (SEQ ID NO:

984) (A02.01)

SQIMSLWGL (SEQ ID NO:

985) (A02.01, A24.02,

B08.01)

SQIMSLWGLV (SEQ ID

NO: 986) (A02.01)

TSKSQIMSL (SEQ ID NO:

987) (B08.01)

NDRG1:ERG
NDRG1:ERG
MSREMQDVDLAEVKPLVE
LLQEFDVQEA (SEQ ID
Prostate Cancer

KGETITGLLQEFDVQ::EAL
NO: 988) (A02.01)

SVVSEDQSLFECAYGTPH

LQEFDVQEAL (SEQ ID

LAKTEMTASSSSDYGQTS

NO: 989) (A02.01)

KMSPRVPQQDW (SEQ ID

NO: 149)

PML:RARA
PML:RARA
VLDMEIGFLRQALCRLRQE

Acute

(exon3:exon3)
EPQSLQAAVRTDGFDEFKV

promyelocytic

RLQDLSSCITQGK:A:IETQS

leukemia

SSSEEIVPSPPSPPPLPRIY

KPCFVCQDKSSGYHYGVS

ACEGCKG (SEQ ID NO:

150)

PML:RARA
PML:RARA
RSSPEQPRPSTSKAVSPPEIL

Acute

(exon6:exon3)
DGPPSPRSPVIGSEVFLPNS

promyelocytic

NHVASGAGEA:A:IETQSSS

leukemia

SEEIVPSPPSPPPLPRIYKP

CFVCQDKSSGYHYGVSAC

EGCKG (SEQ ID NO: 151)

RUNX1
RUNX1
VARFNDLRFVGRSGRGKSF
GPREPRNRT (SEQ ID NO:
AML

(ex5)-
TLTITVFTNPPQVATYHRAI
990) (B07.02)

RUNX1
KITVDGPREPR:N:RTEKHS
RNRTEKHSTM (SEQ ID

T1(ex2)

TMPDSPVDVKTQSRLTPP

NO: 991) (B08.01)

TMPPPPTTQGAPRTSSFTP

TTLTNGT (SEQ ID NO: 152)

TMPRSS2:ERG
TMPRSS2:ERG
MALNS::EALSVVSEDQSLF
ALNSEALSV (SEQ ID NO:
Prostate Cancer

ECAYGTPHLAKTEMTASSS
992) (A02.01)

SDYGQTSKMSPRVPQQDW
ALNSEALSVV (SEQ ID

(SEQ ID NO: 153)
NO: 993) (A02.01)

MALNSEALSV (SEQ ID

NO: 994) (A02.01, B08.01)

TABLE 2A

Amino Acid
Mutation Sequence

Exemplary

Gene
Alteration
Context
Peptides (HLA allele example(s))
Diseases

POINT MUTATIONS¹

AKT1
E17K
MSDVAIVKEGWLH
KYIKTWRPRY (SEQ ID NO:
BRCA, CESC,

KRGKYIKTWRPRYF
1005) (A24.02)
HNSC, LUSC,

LLKNDGTFIGYKERP
WLHKRGKYI (SEQ ID NO:
PRAD, SKCM,

QDVDQREAPLNNFS
1006) (A02.01, B07.02, B08.01)
THCA

VAQCQLMKTER
WLHKRGKYIK (SEQ ID NO:

(SEQ ID NO: 995)
1007) (A03.01)

ANAPC1
T537A
TMLVLEGSGNLVLY
APKPLSKLL (SEQ ID NO: 1008)
GBM, LUSC,

TGVVRVGKVFIPGLP
(B07.02)
PAAD, PRAD,

APSLTMSNTMPRPST
GVSAPKPLSK (SEQ ID NO:
SKCM

PLDGVSAPKPLSKLL
1009) (A03.01)

GSLDEVVLLSPVPEL
VSAPKPLSK (SEQ ID NO: 1010)

RDSSKLEIDSLYNED
(A03.01)

CTFQQLGTYIHSI

(SEQ ID NO: 996)

FGFR3
S249C
HRIGGIKLRHQQWS
CPHRPILQA (SEQ ID NO: 1011)
BLCA, HNSC,

LVMESVVPSDRGNY
(B07.02)
KIRP, LUSC

TCVVENKFGSIRQTY

TLDVLERCPHRPILQ

AGLPANQTAVLGSD

VEFHCKVYSDAQPH

IQWLKHVEVNGSKV

G (SEQ ID NO: 33)

FRG1B
I10T
MREPIYMHSTMVFL
KLSDSRTAL (SEQ ID NO: 1012)
KIRP, PRAD,

PWELHTKKGPSPPE
(A02.01, B07.02, B08.01)
SKCM

QFMAVKLSDSRTAL
KLSDSRTALK (SEQ ID NO:

KSGYGKYLGINSDE
1013) (A03.01)

LVGHSDAIGPREQW
LSDSRTALK (SEQ ID NO: 1014)

EPVFQNGKMALLAS
(A01.01, A03.01)

NSCFIR (SEQ ID NO:
RTALKSGYGK (SEQ ID NO:

997)
1015) (A03.01)

TALKSGYGK (SEQ ID NO:

1016) (A03.01)

FRG1B
L52S
AVKLSDSRIALKSGY
ALSASNSCF (SEQ ID NO: 1017)
GBM, KIRP,

GKYLGINSDELVGH
(A02.01, A24.02, B07.02)
PRAD, SKCM

SDAIGPREQWEPVF
ALSASNSCFI (SEQ ID NO:

QNGKMALSASNSCF
1018) (A02.01)

IRCNEAGDIEAKSKT
FQNGKMALSA (SEQ ID NO:

AGEEEMIKIRSCAEK
1019) (A02.01, B08.01)

ETKKKDDIPEEDKG

(SEQ ID NO: 34)

HER2
L755S
AMPNQAQMRILKET
KVSRENTSPK (SEQ ID NO:
BRCA

(Resistance)
ELRKVKVLGSGAFG
1020) (A03.01)

TVYKGIWIPDGENV

KIPVAIKVSRENTSP

KANKEILDEAYVMA

GVGSPYVSRLLGICL

TSTVQLVTQLMPYG

C (SEQ ID NO: 998)

IDH1
R132G
RVEEFKLKQMWKSP
KPIIIGGHAY (SEQ ID NO:
BLCA, BRCA,

NGTIRNILGGTVFRE
1021) (B07.02)
CRC, GBM,

AIICKNIPRLVSGWV

HNSC, LUAD,

KPIIIGGHAYGDQYR

PAAD, PRAD,

ATDFVVPGPGKVEIT

UCEC

YTPSDGTQKVTYLV

HNFEEGGGVAMGM

(SEQ ID NO: 38)

KRAS
G12C
MTEYKLVVVGACG
KLVVVGACGV (SEQ ID NO:
BRCA, CESC,

VGKSALTIQLIQNHF
154) (A02.01)
CRC, HNSC,

VDEYDPTIEDSYRK
LVVVGACGV (SEQ ID NO:
LUAD, PAAD,

QVVIDGETCLLDILD
155) (A02.01)
UCEC

TAGQE (SEQ ID NO:
VVGACGVGK (SEQ ID NO:

8)
156) (A03.01, A11.01)

VVVGACGVGK (SEQ ID NO:

157) (A03.01)

KRAS
G12D
MTEYKLVVVGADG
VVGADGVGK (SEQ ID NO:
BLCA, BRCA,

VGKSALTIQLIQNHF
158) (A11.01)
CESC, CRC,

VDEYDPTIEDSYRK
VVVGADGVGK (SEQ ID NO:
GBM, HNSC,

QVVIDGETCLLDILD
159) (A11.01)
KIRP, LIHC,

TAGQE (SEQ ID NO:
KLVVVGADGV (SEQ ID NO:
LUAD, PAAD,

9)
160) (A02.01)
SKCM, UCEC

LVVVGADGV (SEQ ID NO:

161) (A02.01)

KRAS
G12V
MTEYKLVVVGAVG
KLVVVGAVGV (SEQ ID NO:
BRCA, CESC,

VGKSALTIQLIQNHF
162) (A02.01)
CRC, LUAD,

VDEYDPTIEDSYRK
LVVVGAVGV (SEQ ID NO:
PAAD, THCA,

QVVIDGETCLLDILD
163) (A02.01)
UCEC

TAGQE (SEQ ID NO:
VVGAVGVGK (SEQ ID NO:

10)
164) (A03.01, A11.01)

VVVGAVGVGK (SEQ ID NO: 5)

(A03.01, A11.01)

KRAS
Q61H
AGGVGKSALTIQLIQ
ILDTAGHEEY (SEQ ID NO:
CRC, LUSC,

NHFVDEYDPTIEDSY
165) (A01.01)
PAAD, SKCM,

RKQVVIDGETCLLDI

UCEC

LDTAGHEEYSAMRD

QYMRTGEGFLCVFA

INNTKSFEDIEHYRE

QIKRVKDSEDVPM

(SEQ ID NO: 11)

KRAS
Q61L
AGGVGKSALTIQLIQ
ILDTAGLEEY (SEQ ID NO: 166)
CRC, GBM,

NHFVDEYDPTIEDSY
(A01.01)
HNSC, LUAD,

RKQVVIDGETCLLDI
LLDILDTAGL (SEQ ID NO: 167)
SKCM, UCEC

LDTAGLEEYSAMRD
(A02.01)

QYMRTGEGFLCVFA

INNTKSFEDIEHYRE

QIKRVKDSEDVPM

(SEQ ID NO: 12)

NRAS
Q61K
AGGVGKSALTIQLIQ
ILDTAGKEEY (SEQ ID NO:
BLCA, CRC,

NHFVDEYDPTIEDSY
168) (A01.01)
LIHC, LUAD,

RKQVVIDGETCLLDI

LUSC, SKCM,

LDTAGKEEYSAMRD

THCA, UCEC

QYMRTGEGFLCVFA

INNSKSFADINLYRE

QIKRVKDSDDVPM

(SEQ ID NO: 13)

NRAS
Q61R
AGGVGKSALTIQLIQ
ILDTAGREEY (SEQ ID NO:
BLCA, CRC,

NHFVDEYDPTIEDSY
169) (A01.01)
LUSC, PAAD,

RKQVVIDGETCLLDI

PRAD, SKCM,

LDTAGREEYSAMRD

THCA, UCEC

QYMRTGEGFLCVFA

INNSKSFADINLYRE

QIKRVKDSDDVPM

(SEQ ID NO: 14)

PIK3CA
E542K
IEEHANWSVSREAG
AISTRDPLSK (SEQ ID NO:
BLCA, BRCA,

FSYSHAGLSNRLAR
1022) (A03.01)
CESC, CRC,

DNELRENDKEQLKA

GBM, HNSC,

ISTRDPLSKITEQEKD

KIRC, KIRP,

FLWSHRHYCVTIPEI

LIHC, LUAD,

LPKLLLSVKWNSRD

LUSC, PRAD,

EVAQMYCLVKDWP

UCEC

P (SEQ ID NO: 48)

PTEN
R130Q
KFNCRVAQYPFEDH
QTGVMICAY (SEQ ID NO:
BRCA, CESC,

NPPQLELIKPFCEDL
1023) (A01.01)
CRC, GBM,

DQWLSEDDNHVAAI

KIRC, LUSC,

HCKAGKGQTGVMIC

UCEC

AYLLHRGKFLKAQE

ALDFYGEVRTRDKK

GVTIPSQRRYVYYY

SY (SEQ ID NO: 52)

RAC1
P29S
MQAIKCVVVGDGA
FSGEYIPTV (SEQ ID NO: 1024)
Melanoma

VGKTCLLISYTTNAF
(A02.01)

SGEYIPTVFDNYSAN
TTNAFSGEY (SEQ ID NO:

VMVDGKPVNLGLW
1025) (A01.01)

DTAGQEDYDRLRPL
YTTNAFSGEY (SEQ ID NO:

SYPQTVGET (SEQ ID
1026) (A01.01)

NO: 53)

SF3B1
K700E
AVCKSKKSWQARH
GLVDEQQEV (SEQ ID NO:
AML associated

TGIKIVQQIAILMGC
1027) (A02.01)
with MDS;

AILPHLRSLVEIIEHG

Chronic

LVDEQQEVRTISALA

lymphocytic

IAALAEAATPYGIES

leukaemia-small

FDSVLKPLWKGIRQ

lymphocytic

HRGKGLAAFLKAI

lymphoma;

(SEQ ID NO: 999)

Myelodysplastic

syndrome; AML;

Luminal NS

carcinoma of

breast; Chronic

myeloid

leukaemia; Ductal

carcinoma of

pancreas; Chronic

myelomonocytic

leukaemia;

Chronic

lymphocytic

leukaemia-small

lymphocytic

lymphoma;

Myelofibrosis;

Myelodysplastic

syndrome; PRAD;

Essential

thrombocythaemia;

Medullomyoblastoma

SPOP
F133L
YLSLYLLLVSCPKSE
FVQGKDWGL (SEQ ID NO:
PRAD

VRAKFKFSILNAKGE
1028) (A02.01, B08.01)

ETKAMESQRAYRFV

QGKDWGLKKFIRRD

FLLDEANGLLPDDK

LTLFCEVSVVQDSV

NISGQNTMNMVKVP

E (SEQ ID NO: 1000)

SPOP
F133V
YLSLYLLLVSCPKSE
FVQGKDWGV (SEQ ID NO:
PRAD

VRAKFKFSILNAKGE
1029) (A02.01)

ETKAMESQRAYRFV

QGKDWGVKKFIRRD

FLLDEANGLLPDDK

LTLFCEVSVVQDSV

NISGQNTMNMVKVP

E (SEQ ID NO: 1001)

TP53
G245S
IRVEGNLRVEYLDD
CMGSMNRRPI (SEQ ID NO:
BLCA, BRCA,

RNTFRHSVVVPYEPP
1030) (A02.01, B08.01)
CRC, GBM,

EVGSDCTTIHYNYM
GSMNRRPIL (SEQ ID NO: 1031)
HNSC, LUSC,

CNSSCMGSMNRRPI
(B08.01)
PAAD, PRAD

LTIITLEDSSGNLLGR
MGSMNRRPI (SEQ ID NO:

NSFEVRVCACPGRD
1032) (B08.01)

RRTEEENLRKKGEP
MGSMNRRPIL (SEQ ID NO:

(SEQ ID NO: 54)
1033) (B08.01)

SMNRRPILTI (SEQ ID NO:

1034) (A02.01, A24.02, B08.01)

TP53
R248Q
EGNLRVEYLDDRNT
CMGGMNQRPI (SEQ ID NO:
BLCA, BRCA,

FRHSVVVPYEPPEV
1035) (A02.01, B08.01)
CRC, GBM,

GSDCTTIHYNYMCN
GMNQRPILTI (SEQ ID NO:
HNSC, KIRC,

SSCMGGMNQRPILTI
1036) (A02.01, B08.01)
LIHC, LUSC,

ITLEDSSGNLLGRNS
NQRPILTII (SEQ ID NO: 1037)
PAAD, PRAD,

FEVRVCACPGRDRR
(A02.01, B08.01)
UCEC

TEEENLRKKGEPHH

E (SEQ ID NO: 56)

TP53
R248W
EGNLRVEYLDDRNT
CMGGMNWRPI (SEQ ID NO:
BLCA, BRCA,

FRHSVVVPYEPPEV
1038) (A02.01, A24.02, B08.01)
CRC, GBM,

GSDCTTIHYNYMCN
GMNWRPILTI (SEQ ID NO:
HNSC, LIHC,

SSCMGGMNWRPILT
1039) (A02.01, B08.01)
LUSC, PAAD,

IITLEDSSGNLLGRNS
MNWRPILTI (SEQ ID NO: 1040)
SKCM, UCEC

FEVRVCACPGRDRR
(A02.01, A24.02, B08.01)

TEEENLRKKGEPHH
MNWRPILTII (SEQ ID NO:

E (SEQ ID NO: 57)
1041) (A02.01, A24.02)

TP53
R273C
PEVGSDCTTIHYNY
NSFEVCVCA (SEQ ID NO:
BLCA, BRCA,

MCNSSCMGGMNRR
1042) (A02.01)
CRC, GBM,

PILTIITLEDSSGNLL

HNSC, LUSC,

GRNSFEVCVCACPG

PAAD, UCEC

RDRRTEEENLRKKG

EPHHELPPGSTKRAL

PNNTSSSPQPKKKPL

(SEQ ID NO: 58)

TP53
R273H
PEVGSDCTTIHYNY
NSFEVHVCA (SEQ ID NO:
BRCA, CRC,

MCNSSCMGGMNRR
1043) (A02.01)
GBM, HNSC,

PILTIITLEDSSGNLL

LIHC, LUSC,

GRNSFEVHVCACPG

PAAD, UCEC

RDRRTEEENLRKKG

EPHHELPPGSTKRAL

PNNTSSSPQPKKKPL

(SEQ ID NO: 1002)

TP53
Y220C
TEVVRRCPEIHERCS
VVPCEPPEV (SEQ ID NO: 1044)
BLCA, BRCA,

DSDGLAPPQHLIRVE
(A02.01)
GBM, HNSC,

GNLRVEYLDDRNTF
VVVPCEPPEV (SEQ ID NO:
LIHC, LUAD,

RHSVVVPCEPPEVGS
1045) (A02.01)
LUSC, PAAD,

DCTTIHYNYMCNSS

SKCM, UCEC

CMGGMNRRPILTIIT

LEDSSGNLLGRNSF

(SEQ ID NO: 1003)

TABLE 2B

MSI-ASSOCIATED FRAMESHIFTS¹

MSH6
F1088fs; +1
YNFDKNYKDWQSA
ILLPEDTPPL (SEQ ID NO: 1046)
MSI+ CRC, MSI+

VECIAVLDVLLCLA
(A02.01)
Uterine/Endometrium

NYSRGGDGPMCRPV
LLPEDTPPL (SEQ ID NO: 1047)
Cancer, MSI+

ILLPEDTPPLLRA
(A02.01)
Stomach Cancer,

(SEQ ID NO: 1004)

Lynch syndrome

TABLE 2C

FRAMESHIFT¹

Amino Acid
Mutation Sequence

Exemplary

Gene
Alteration
Context
Peptides (HLA allele example(s))
Diseases

APC
F1354fs

AKFQQCHSTLEPNP

APFRVNHAV (SEQ ID NO:
CRC, LUAD,

ADCRVLVYLQNQPG

1048)(B07.02)
UCEC, STAD

TKLLNFLQERNLPPK

CLADVLLSV (SEQ ID NO:

VVLRHPKVHLNTMF

1049)(A02.01)

RRPHSCLADVLLSV

FLQERNLPPK (SEQ ID NO:

HLIVLRVVRLPAPFR

1050)(A03.01)

VNHAVEW* (SEQ ID
HLIVLRVVRL (SEQ ID NO:

NO: 95)
1051)(A02.01, B08.01)

HPKVHLNTM (SEQ ID NO:

1052)(B07.02, B08.01)

HPKVHLNTMF (SEQ ID NO:

1053)(B07.02, B08.01)

KVHLNTMFR (SEQ ID NO:

1054)(A03.01)

KVHLNTMFRR (SEQ ID NO:

1055)(A03.01)

LPAPFRVNHA (SEQ ID NO:

1056)(B07.02)

MFRRPHSCL (SEQ ID NO:

1057)(B07.02, B08.01)

MFRRPHSCLA (SEQ ID NO:

1058)(B08.01)

NTMFRRPHSC (SEQ ID NO:

1059)(B08.01)

RPHSCLADV (SEQ ID NO:

1060)(B07.02)

RPHSCLADVL (SEQ ID NO:

1061)(B07.02)

RVVRLPAPFR (SEQ ID NO:

1062)(A03.01)

SVHLIVLRV (SEQ ID NO: 1063)

(A02.01)

TMFRRPHSC (SEQ ID NO:

1064)(B08.01)

TMFRRPHSCL (SEQ ID NO:

1065)(A02.01, B08.01)

VLLSVHLIV (SEQ ID NO: 1066)

(A02.01)

VLLSVHLIVL (SEQ ID NO:

1067)(A02.01)

VLRVVRLPA (SEQ ID NO:

1068)(B08.01)

VVRLPAPFR (SEQ ID NO: 1069)

(A03.01)

A subset of peptides from Table 1 (n=562) were synthesized and their affinity for their given HLA class I molecule was measured as described. The values are shown in Table 3. These data show a strong correlation between prediction and measurement (dotted line represents best fit, R²=0.45), demonstrating the value of the predictions. However, the outliers demonstrate the importance of these measurements. Thick vertical and horizontal lines are shown at 500 nM for the predicted affinity and observed affinity, respectively. 500 nM is commonly accepted in the field as the maximum affinity for an epitope that is a “weak binder” to HLA class I. Therefore, the points in the lower right quadrant (prediction greater than 500 nM, measurement less than 500 nM) are epitopes that were considered very weak binders but were observed to bind within an acceptable range. Epitopes in this quadrant (n=75) represent 30.5% of epitopes not considered to be binders by prediction (combination of bottom right and top right quadrants, n=246).

TABLE 3

Observed

Predicted
Affinity

SEQ ID
affinity
(IC50;
Stability

Mutation
Allele
Peptide
NO:
(IC50; (nM))
(nM))
(T1/2 (h))

ABL1, M351T
A02.01
TQISSATEYL
208
2921.0
2644.0
0

ABL1, T315I
A02.01
YIIIEFMTYG
214
3502.0
186.0
0

ABL1, T315I
A02.01
IIIEFMTYG
212
1991.0
779.0
0

ABL1, T315I
A02.01
IIIEFMTYGN
213
16793.0
1551.0
0

ABL1, T315I
A02.01
IIEFMTYGNL
211
2134.0
9702.0
0

ABL1, Y253H
A02.01
KLGGGQHGEV
216
1705.0
387.0
0.4

AKT1, E17K
B08.01
WLHKRGKYI
1006
47.0
417.0
1.3

AKT1, E17K
A02.01
WLHKRGKYI
1006
4972.0
1250.0
1.2

AKT1, E17K
B07.02
WLHKRGKYI
1006
7185.0
2648.0
0

ALK, G1269A
A02.01
RVAKIADFGM
218
5258.0
125.0
0.5

ALK, G1269A
B07.02
RVAKIADFGM
218
7260.0
9723.0
0.2

ALK, L1196M
A02.01
SLPRFILMEL
226
94.0
26.0
0.5

ALK, L1196M
A02.01
ILMELMAGG
220
192.0
223.0
0.5

ALK, L1196M
A02.01
LMELMAGGDL
222
5617.0
311.0
8.9

ALK, L1196M
A02.01
LQSLPRFILM
225
2519.0
413.0
0

ALK, L1196M
B07.02
SLPRFILMEL
226
17.0
583.0
0.4

ALK, L1196M
B08.01
LQSLPRFILM
225
1288.0
1547.0
0

ALK, L1196M
A02.01
FILMELMAGG
219
189.0
1580.0
0

ALK, L1196M
B08.01
SLPRFILMEL
226
686.0
1762.0
0

ALK, L1196M
A24.02
SLPRFILMEL
226
5143.0
2774.0
0.2

ALK, L1196M
A02.01
ILMELMAGGD
221
5761.0
3451.0
0

APC, AVEW
A02.01
VLLSVHLIV
1066
36.0
72.0
11

(SEQ ID NO:

1130)

APC, AVEW
A02.01
CLADVLLSV
1049
5.0
219.0
24

(SEQ ID NO:

1130)

APC, VHPA
A02.01
KVLQMDFLV
479
25.0
11.0
6.4

(SEQ ID NO:

1131)

APC, VHPA
A02.01
LQMDFLVEIPA
481
26.0
68.0
1.5

(SEQ ID NO:

1131)

β2M, . . . MPAV
A03.01
TTLNSPPLKK
651
62.5
14.3
not

(SEQ ID NO:

measured

1132)

β2M, . . . MPAV
A03.01
TTLNSPPLK
650
165.4
9.8
not

(SEQ ID NO:

measured

1132)

β2M, . . . MPAV
A03.01
TLNSPPLKK
649
27.5
5.4
not

(SEQ ID NO:

measured

1132)

β2M, . . . MPAV
A03.01
CTTLNSPPLK
646
225.3
63.6
not

(SEQ ID NO:

measured

1132)

β2M, . . . MPAV
B08.01
CLSARTGLSI
645
1106.6
149.6
not

(SEQ ID NO:

measured

1132)

β2M, . . . MPAV
A02.01
GLSISCTTL
647
669.0
114.5
not

(SEQ ID NO:

measured

1132)

β2M, . . . SIRH
A03.01
LTSSSREWK
1070
413.9
117.8
not

(SEQ ID NO:

measured

1133)

β2M, . . . SIRH
A03.01
LLTSSSREWK
1071
206.1
1769.8
not

(SEQ ID NO:

measured

1133)

β2M, . . . SIRH
B07.02
YPAYSKDSGL
1072
41.1
79.5
not

(SEQ ID NO:

measured

1133)

β2M, . . . SIRH
B08.01
EWKVKFPEL
1073
488.7
538.4
not

(SEQ ID NO:

measured

1133)

β2M, . . . SIRH
A24.02
KFPELLCVW
1074
83.7
13.7
not

(SEQ ID NO:

measured

1133)

β2M, . . . SQIS
B08.01
LQRFRFTHV
652
55.5
37.3
not

(SEQ ID NO:

measured

1134)

β2M, . . . SQIS
A24.02
RLSSVLQRF
654
288.9
28.2
not

(SEQ ID NO:

measured

1134)

β2M, . . . SQIS
A02.01
VLQRFRFTHV
656
163.4
106.7
not

(SEQ ID NO:

measured

1134)

β2M, . . . SQIS
B08.01
VLQRFRFTHV
656
264.1
480.1
not

(SEQ ID NO:

measured

1134)

β2M, . . . SQIS
A03.01
RLSSVLQRFR
655
168.1
12.5
not

(SEQ ID NO:

measured

1134)

BCR: ABL
B08.01
LTINKEEAL
964
4972.0
895.0
0

(e13a2, aka b2a2)

BCR: ABL
A02.01
LTINKEEAL
964
12671.0
4413.0
0

(e13a2, aka b2a2)

BRAF, V600E
A02.01
LATEKSRWSG
228
39130.0
23337.0
0

BRAF, V600E
B08.01
LATEKSRWS
227
24674.0
36995.0
0

BRAF, V600E
B08.01
LATEKSRWSG
228
13368.0
46582.0
0

BRAF, V600E
A02.01
LATEKSRWS
227
39109.0
60997.0
0

BTK, C481S
A02.01
SLLNYLREM
173
48.0
87.0
3

BTK, C481S
A02.01
MANGSLLNYL
172
2979.0
1082.0
0

BTK, C481S
B07.02
SLLNYLREM
173
6544.0
1110.0
0

BTK, C481S
B08.01
SLLNYLREM
173
1091.0
1230.0
0

BTK, C481S
A02.01
YMANGSLLN
174
7856.0
4444.0
0

BTK, C481S
B07.02
MANGSLLNYL
172
8921.0
17715.0
0

BTK, C481S
B08.01
MANGSLLNYL
172
7639.0
19853.0
0

BTK, C481S
A03.01
MANGSLLNY
171
1030.3
35.6
not

measured

BTK, C481S
A01.01
MANGSLLNY
171
285.7
439.0
not

measured

BTK, C481S
A24.02
EYMANGSLL
170
213.2
5.0
not

measured

BTK, C481S
A01.01
YMANGSLLNY
175
95.7
13.2
not

measured

BTK, C481S
A03.01
YMANGSLLNY
175
109.4
95.9
not

measured

C11orf95: RELA
A02.01
ELFPLIFPA
967
13.0
13.0
5.1

C11orf95: RELA
A24.02
KGPELFPLI
968
909.0
14.0
1.7

C11orf95: RELA
A02.01
KGPELFPLI
968
6840.0
101.0
0.3

C11orf95: RELA
B08.01
ELFPLIFPA
967
7316.0
449.0
0

C15ORF40(+1)
A02.01
KLFSCLSFL
344
6.0
6.0
14.3

C15ORF40(+1)
A03.01
KLFSCLSFL
344
1488.0
308.0
0.8

C15ORF40(+1)
A03.01
SLQPPPPGFK
352
26.3
19.0
not

measured

C15ORF40(+1)
A03.01
LFFFFFETK
347
658.4
413.3
not

measured

C15ORF40(+1)
A02.01
ALFFFFFET
334
28.9
470.7
not

measured

C15ORF40(+1)
A03.01
ALFFFFFETK
335
31.5
216.4
not

measured

C15ORF40(+1)
A02.01
FFFETKSCSV
340
754.5
61.2
not

measured

C15ORF40(+1)
B08.01
FFETKSCSV
339
807.6
7.6
not

measured

C15ORF40(+1)
A01.01
LSFLSSWDY
348
211.1
52.9
not

measured

C15ORF40(+1)
A02.01
FLSSWDYRRM
342
62.2
323.5
not

measured

C15ORF40(+1)
A03.01
LSFLSSWDYR
349
508.7
100.9
not

measured

C15ORF40(+1)
A02.01
FKLFSCLSFL
341
9.9
662.9
not

measured

C15ORF40(+1)
A02.01
VQWRSLGSL
353
986.0
4733.2
not

measured

C15ORF40(+1)
A02.01
KLFSCLSFLS
345
65.1
0.6
not

measured

C15ORF40(+1)
A03.01
KLFSCLSFLS
345
805.1
104.0
not

measured

C15ORF40(+1)
A02.01
AQAGVQWRSL
336
630.2
670.0
not

measured

C15ORF40(+1)
A24.02
RRMPPCLANF
351
253.0
141.1
not

measured

C15ORF40(+1)
A03.01
CLSFLSSWDY
338
890.5
2705.8
not

measured

C15ORF40(+1)
A24.02
GFKLFSCLSF
343
387.4
643.0
not

measured

C15ORF40(+1)
A24.02
RMPPCLANF
350
34.4
8.7
not

measured

C15ORF40(+1)
A03.01
CLANFCIFNR
337
575.4
221.8
not

measured

C15ORF40(+1)
A01.01
CLSFLSSWDY
338
538.7
987.3
not

measured

CNOT1(+1)
A02.01
SVCFFFFSV
356
27.0
175.0
9.4

CNOT1(+1)
B08.01
SVCFFFFSV
356
4940.0
10599.0
0

CNOT1(+1)
A02.01
MSVCFFFFSV
355
131.0
1706.4
not

measured

CNOT1(+1)
A02.01
FFFSVIFST
354
608.9
4556.0
not

measured

CNOT1(−1)
A01.01
MSVCFFFFCY
359
310.4
4369.3
not

measured

CNOT1(−1)
A02.01
SVCFFFFCYI
360
237.2
519.8
not

measured

CNOT1(−1)
A24.02
FFCYILNTMF
358
583.4
73.4
not

measured

EGFR, T790M
A02.01
MQLMPFGCLL
184
21.0
20.0
0.4

EGFR, T790M
A02.01
MQLMPFGCL
183
842.0
166.0
0.4

EGFR, T790M
A02.01
LIMQLMPFGC
181
1984.0
177.0
0.4

EGFR, T790M
A02.01
QLIMQLMPF
185
2511.0
227.0
0.3

EGFR, T790M
B08.01
QLIMQLMPF
185
891.0
388.0
0

EGFR, T790M
B08.01
IMQLMPFGCL
179
1302.0
548.0
0

EGFR, T790M
A02.01
CLTSTVQLIM
177
3465.0
716.0
0

EGFR, T790M
A02.01
IMQLMPFGCL
179
143.0
837.0
0.5

EGFR, T790M
A02.01
IMQLMPFGC
178
1123.0
1607.0
0.4

EGFR, T790M
B07.02
MQLMPFGCL
183
10169.0
2270.0
0

EGFR, T790M
A24.02
QLIMQLMPF
185
3209.0
2389.0
0.4

EGFR, T790M
A02.01
LIMQLMPFG
180
4961.0
3513.0
0

EGFR, T790M
A24.02
VQLIMQLMPF
188
1455.0
4559.0
0

EGFR, T790M
A02.01
VQLIMQLMPF
188
4464.0
5492.0
0

EGFR, T790M
A02.01
QLIMQLMPFG
186
5751.0
5926.0
0

EGFR, T790M
B08.01
MQLMPFGCL
183
1105.0
7045.0
0

EGFR, T790M
A02.01
STVQLIMQL
187
2151.0
8537.0
0

EGFR, T790M
A01.01
CLTSTVQLIM
177
2998.0
11036.0
0

EGFR, T790M
B08.01
MQLMPFGCLL
184
970.0
14056.0
0

EGFR, T790M
B08.01
VQLIMQLMPF
188
3370.0
17898.0
0

EGFR, T790M
A24.02
IMQLMPFGCL
179
4394.0
18102.0
0

EGFR, T790M
A24.02
MQLMPFGCLL
184
4168.0
23572.0
0

EGFR, T790M
A01.01
LTSTVQLIM
182
1000.7
2891.1
not

measured

EGFR: SEPT14
B08.01
QLQDKFEHL
973
917.0
989.0
0

EGFR: SEPT14
A02.01
QLQDKFEHL
973
422.0
1155.0
0.6

EGFR: SEPT14
A02.01
YLVIQLQDKF
975
9963.0
2057.0
0

EGFR: SEPT14
A24.02
YLVIQLQDKF
975
9508.0
2152.0
2.6

EGFR: SEPT14
A02.01
IQLQDKFEHL
972
820.0
4265.0
0.2

EGFR: SEPT14
B08.01
IQLQDKFEHL
972
4278.0
10247.0
0

EGFRvIII
A02.01
ALEEKKGNYV
971
2445.0
141.0
0

(internal deletion)

EIF2B3(−1)
A02.01
KQWSSVTSL
361
54.4
26.5
not

measured

EML4: ALK
B08.01
QVYRRKHQEL
976
194.0
160.0
0

EPHB2(−1)
A02.01
ILIRKAMTV
363
38.2
19.5
not

measured

ESR1, D538G
A24.02
PLYGLLLEML
234
1519.0
444.0
6.3

ESR1, D538G
A02.01
GLLLEMLDA
230
705.0
558.0
0.4

ESR1, D538G
A02.01
PLYGLLLEM
233
349.0
640.0
0.7

ESR1, D538G
A24.02
VVPLYGLLL
236
2965.0
658.0
0.8

ESR1, D538G
A02.01
PLYGLLLEML
234
542.0
797.0
0

ESR1, D538G
A02.01
VVPLYGLLL
236
4432.0
1039.0
0.6

ESR1, D538G
A02.01
NVVPLYGLL
232
4835.0
10471.0
0

ESR1, D538G
B07.02
VPLYGLLLEM
235
145.1
27.9
not

measured

ESR1, D538G
A24.02
LYGLLLEML
231
218.3
0.8
not

measured

ESR1, S463P
A02.01
FLPSTLKSL
237
71.0
21.0
2.1

ESR1, S463P
A02.01
GVYTFLPST
238
307.0
779.0
1.3

ESR1, S463P
A24.02
FLPSTLKSL
237
10723.0
995.0
1

ESR1, S463P
A02.01
GVYTFLPSTL
239
248.0
1197.0
0.4

ESR1, S463P
B08.01
FLPSTLKSL
237
2314.0
1968.0
0

ESR1, S463P
A24.02
GVYTFLPSTL
239
954.0
7696.0
0

ESR1, Y537C
A02.01
PLCDLLLEM
245
1067.0
602.0
0.8

ESR1, Y537C
A02.01
VVPLCDLLL
248
5533.0
1200.0
0

ESR1, Y537C
A02.01
NVVPLCDLLL
244
1964.0
1373.0
0

ESR1, Y537C
A02.01
PLCDLLLEML
246
1320.0
2008.0
0.9

ESR1, Y537C
A02.01
NVVPLCDLL
243
3473.0
3027.0
0

ESR1, Y537C
A24.02
VVPLCDLLL
248
7992.0
3888.0
0.4

ESR1, Y537N
A02.01
PLNDLLLEM
251
1062.0
151.0
4.2

ESR1, Y537N
A02.01
NVVPLNDLL
249
4725.0
2900.0
0

ESR1, Y537N
A02.01
NVVPLNDLLL
250
2606.0
4190.0
0

ESR1, Y537N
A02.01
PLNDLLLEML
252
1741.0
11957.0
0

ESR1, Y537S
A02.01
PLSDLLLEM
256
713.0
404.0
2.7

ESR1, Y537S
A02.01
NVVPLSDLLL
255
2510.0
741.0
0

ESR1, Y537S
A02.01
NVVPLSDLL
254
4259.0
916.0
0

ESR1, Y537S
A02.01
VVPLSDLLL
259
8320.0
2551.0
0

E5R1, Y537S
A02.01
PLSDLLLEML
257
1138.0
6469.0
0

ESR1, Y537S
A24.02
VVPLSDLLL
259
8463.0
8252.0
0.5

FAM111B(−1)
A03.01
RMKVPLMK
364
58.9
33.0
not

measured

FGFR3, S249C
A02.01
YTLDVLERC
261
3309.0
1764.0
6.6

FGFR3, S249C
B08.01
VLERCPHRPI
260
3629.0
7223.0
0

FGFR3, S249C
A02.01
VLERCPHRPI
260
4505.0
15321.0
0

FGFR3: TACC3
A02.01
VLTVTSTDV
979
1255.0
295.0
1.1

FRG1B, I1OT
B07.02
KLSDSRTAL
1012
225.0
9.0
6.8

FRG1B, I1OT
A02.01
KLSDSRTAL
1012
275.0
111.0
2.8

FRG1B, I1OT
B08.01
KLSDSRTAL
1012
3276.0
122.0
0

FRG1B, L52S
A02.01
ALSASNSCFI
1018
327.0
226.0
0.8

FRG1B, L52S
B08.01
FQNGKMALSA
1019
7796.0
425.0
0

FRG1B, L52S
B07.02
ALSASNSCF
1017
13989.0
684.0
0

FRG1B, L52S
A02.01
ALSASNSCF
1017
7913.0
728.0
0.3

FRG1B, L52S
A02.01
FQNGKMALS
262
2305.0
3276.0
0

FRG1B, L52S
A02.01
FQNGKMALSA
1019
1205.0
6158.0
0

FRG1B, L52S
A24.02
ALSASNSCF
1017
9672.0
16338.0
0.2

GATA3 . . . CSNH
B08.01
FLKAESKIM
1075
263.4
21.9
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
LQHGHRHGL
1076
693.0
550.4
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
EPHLALQPL
1077
106.6
17.0
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
RPLQTHVLPE
706
968.0
2534.4
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
FATLQRSSL
1078
138.0
26.6
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
MFATLQRSSL
1079
1285.0
266.9
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A24.02
MFLKAESKI
1080
1065.7
332.1
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
FATLQRSSL
1078
261.9
14.0
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
MLTGPPARV
1081
145.4
10.6
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
EPHLALQPL
1077
1128.3
12.4
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
GPPARVPAV
1082
297.6
221.2
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
MFATLQRSSL
1079
220.5
53.4
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
ALQPLQPHA
1083
644.4
603.9
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A03.01
VLWTTPPLQH
707
962.3
16.0
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
VLPEPHLAL
1084
140.7
16.0
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
HVLPEPHLAL
705
1057.2
1332.6
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A03.01
YMFLKAESK
1085
53.1
79.8
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
VPAVPFDLHF
1086
1996.2
2114.2
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
AIQPVLWTT
1087
229.3
8.1
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
TLQRSSLWCL
1088
319.2
117.7
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A03.01
KIMFATLQR
1089
62.5
2.5
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
QPVLWTTPPL
1090
54.4
109.3
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
ESKIMFATL
1091
253.7
17.7
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
IMKPKRDGYM
1092
342.1
33.2
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
KPKRDGYMF
1093
109.7
28.2
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
FLKAESKIMF
1094
1539.9
82.3
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B07.02
KPKRDGYMFL
1095
32.5
98.1
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
B08.01
LHFCRSSIM
1096
2141.2
118.7
not

(SEQ ID NO:

measured

1135)

GATA3 . . . CSNH
A02.01
SMLTGPPARV
6
57.0
15.0
21.7

(SEQ ID NO:

1135)

GATA3 . . . CSNH
B08.01
YMFLKAESKI
1097
606.0
32.0
0.4

(SEQ ID NO:

1135)

GATA3 . . . CSNH
A02.01
YMFLKAESKI
1097
163.0
166.0
0.6

(SEQ ID NO:

1135)

GATA3 . . . CSNH
A03.01
YMFLKAESKI
1097
1338.0
21111.0
0

(SEQ ID NO:

1135)

GATA3 YHEA
A02.01
FLQEQYHEA
710
7.0
11.0
9.5

(SEQ ID NO:

1136)

GATA3 YHEA
B08.01
FLQEQYHEA
710
1222.0
2285.0
0

(SEQ ID NO:

1136)

GBP3(−1)
B08.01
TLKKKPRDI
365
286.3
3.3
not

measured

HER2,
A02.01
VMAYVMAGV
1098
6.0
2.0
16.5

G776insYVMA

(SEQ ID NO:

1137)

HER2,
A02.01
YVMAYVMAGV
1099
5.0
57.0
20.9

G776insYVMA

(SEQ ID NO:

1137)

HER2,
B07.02
YVMAYVMAG
1100
6910.0
170.0
0

G776insYVMA

(SEQ ID NO:

1137)

HER2,
B08.01
YVMAYVMAGV
1099
721.0
353.0
0

G776insYVMA

(SEQ ID NO:

1137)

HER2,
A02.01
YVMAYVMAG
1100
841.0
11535.0
2.5

G776insYVMA

(SEQ ID NO:

1137)

HER2,
B08.01
YVMAYVMAG
1100
836.0
19413.0
1.2

G776insYVMA

(SEQ ID NO:

1137)

HER2,
B07.02
YVMAYVMAGV
1099
11445.0
52630.0
0

G776insYVMA

(SEQ ID NO:

1137)

HER2, L7555
A03.01
KVSRENTSPK
1020
66.0
7.0
13.5

HER2, V777L
A02.01
VMAGLGSPYV
263
20.0
102.0
2.9

HER2, V777L
A03.01
VMAGLGSPYV
263
3951.0
11222.0
0

JAK1(−1)
A02.01
SLMPAHWSI
1101
4.0
48.0
5

JAK1(−1)
A02.01
LSLMPAHWSI
1102
21.0
164.0
0.4

JAK1(−1)
B08.01
SLMPAHWSI
1101
282.0
177.0
0

JAK1(−1)
A02.01
FQMQPLSLM
1103
33.0
553.0
0.3

JAK1(−1)
A24.02
SLMPAHWSI
1101
194.0
633.0
0.4

JAK1(−1)
B08.01
LSLMPAHWSI
1102
1914.0
860.0
0

JAK1(−1)
B07.02
SLMPAHWSI
1101
3907.0
1040.0
0

JAK1(−1)
B08.01
FQMQPLSLM
1103
2261.0
6714.0
0

JAK1(−1)
B07.02
FQMQPLSLM
1103
3458.0
10207.0
0

JAK1(−1)
A24.02
LSLMPAHWSI
1102
2125.0
12398.0
0

JAK1(−1)
A24.02
FQMQPLSLM
1103
4021.0
14612.0
0

KIT, T670I
A02.01
VIIEYCCYG
270
4225.0
191.0
0.5

KIT, T670I
A02.01
IIEYCCYGDL
268
3918.0
7310.0
0

KIT, T670I
A02.01
TIGGPTLVII
269
5425.0
10685.0
0

KIT, V654A
A02.01
YLGNHMNIA
274
92.0
117.0
0.6

KIT, V654A
A02.01
MNIANLLGA
273
4522.0
128.0
0.3

KIT, V654A
A02.01
HMNIANLLGA
271
294.0
430.0
0

KIT, V654A
B08.01
YLGNHMNIA
274
2480.0
872.0
0

KIT, V654A
A02.01
YLGNHMNIAN
275
7103.0
1342.0
0

KIT, V654A
A02.01
IANLLGACTI
272
11214.0
6417.0
0

KRAS, G12C
A02.01
KLVVVGACGV
154
204.0
150.0
1

KRAS, G12C
A02.01
LVVVGACGV
155
658.0
1213.0
0.6

KRAS, G12C
A03.01
VVVGACGVGK
157
300.7
1.6
not

measured

KRAS, G12C
A03.01
VVGACGVGK
156
182.0
4.1
not

measured

KRAS, G12D
A02.01
KLVVVGADGV
160
361.0
184.0
0.9

KRAS, G12D
A02.01
LVVVGADGV
161
2120.0
1192.0
0

KRAS, G12V
A02.01
KLVVVGAVGV
162
163.0
96.0
0.9

KRAS, G12V
A02.01
LVVVGAVGV
163
453.0
975.0
0.6

KRAS, G12V
A03.01
VVGAVGVGK
164
168.9
1.9
not

measured

KRAS, Q61H
A01.01
ILDTAGHEEY
165
131.8
64.2
not

measured

KRAS, Q61L
A01.01
ILDTAGLEEY
166
65.9
8.6
not

measured

KRAS, Q61L
A02.01
LLDILDTAGL
167
113.4
715.7
not

measured

LMAN1(+1)
B07.02
GPPRPPRAAC
373
69.3
48.6
not

measured

LMAN1(+1)
B07.02
PPRPPRAAC
374
263.7
32.8
not

measured

LMAN1(−1)
B08.01
SLRRKYLRV
375
28.0
0.4
not

measured

MEK, C121S
A02.01
VLHESNSPYI
277
189.0
131.0
1.9

MEK, P124L
A02.01
VLHECNSLYI
285
67.0
10.0
5.1

MEK, P124L
A02.01
SLYIVGFYGA
283
104.0
390.0
0.4

MEK, P124L
A02.01
SLYIVGFYG
282
2987.0
1063.0
0

MEK, P124L
A02.01
LQVLHECNSL
278
5803.0
4723.0
0

MEK, P124L
A02.01
QVLHECNSL
281
8695.0
7774.0
0.5

MEK, P124L
A03.01
VLHECNSLYI
285
4733.0
10500.0
0

MEK, P124L
B08.01
QVLHECNSL
281
6854.0
14532.0
0

MEK, P124L
B08.01
LQVLHECNSL
278
2782.0
19316.0
0

MLL2, . . . LSPH
A02.01
LLQVTQTSFA
1104
1935.0
676.9
not

(SEQ ID NO:

measured

1138)

MLL2, . . . LSPH
A02.01
RLWHLLLQV
1105
8.3
1.3
not

(SEQ ID NO:

measured

1138)

MLL2, . . . LSPH
A02.01
LQVTQTSFAL
1106
1147.4
718.3
not

(SEQ ID NO:

measured

1138)

MLL2, . . . LSPH
A02.01
RLWHLLLQVT
1107
140.8
50.9
not

(SEQ ID NO:

measured

1138)

MLL2, . . . LSPH
A02.01
ALAPTLTHM
1108
98.4
59.0
not

(SEQ ID NO:

measured

1138)

MLL2, . . . LSPH
A02.01
ALAPTLTHML
1109
66.4
39.0
not

(SEQ ID NO:

measured

1138)

MLL2, CRLS
B08.01
SLGNHLCPL
1110
136.0
6.0
0.5

(SEQ ID NO:

1139)

MLL2, CRLS
A02.01
SLGNHLCPL
1110
28.0
18.0
3.5

(SEQ ID NO:

1139)

MLL2, CRLS
B07.02
SLGNHLCPL
1110
3967.0
2590.0
0

(SEQ ID NO:

1139)

MSH3(−1)
A02.01
LLALWECSL
385
46.0
15.0
4

MSH3(−1)
A02.01
FLLALWECSL
381
17.0
114.0
10.8

MSH3(−1)
B08.01
LLALWECSL
385
1454.0
154.0
0

MSH3(−1)
B08.01
FLLALWECSL
381
671.0
13100.0
0

MSH3(−1)
A02.01
LIVSRTLLL
383
755.0
173.5
not

measured

MSH3(−1)
A02.01
LIVSRTLLLV
384
146.6
10920.6
not

measured

MSH3(−1)
B08.01
LIVSRTLLL
383
270.7
881.7
not

measured

MSH3(−1)
A02.01
IVSRTLLLV
382
166.2
12.7
not

measured

MSH3(−1)
B08.01
SLPQARLCLI
389
632.3
4313.9
not

measured

MSH3(−1)
B08.01
CLIVSRTLLL
379
835.7
1100.4
not

measured

MSH3(−1)
B08.01
LPQARLCLI
386
136.5
15.0
not

measured

MSH3(−1)
A02.01
SLPQARLCLI
389
782.4
112.9
not

measured

MSH3(−1)
A02.01
CLIVSRTLLL
379
560.5
2005.1
not

measured

MSH3(−1)
A02.01
FLLALWECS
380
686.6
93.2
not

measured

MSH3(−1)
A02.01
FLLALWECSL
381
16.6
0.9
not

measured

MSH3(−1)
A02.01
LLALWECSL
385
46.1
12.5
not

measured

MSH3(−1)
B07.02
LPQARLCLI
386
134.6
72.6
not

measured

MSH3(−1)
B08.01
CLIVSRTLL
378
915.0
126.7
not

measured

MSH3(−1)
A02.01
ALWECSLPQA
377
24.6
9.0
not

measured

MSH3(−1)
B08.01
LPQARLCLIV
387
591.4
152.1
not

measured

MSH6(+1)
A02.01
LLPEDTPPL
1047
8.9
2.8
not

measured

MSH6(+1)
A02.01
ILLPEDTPPL
1046
16.3
6.7
not

measured

MYC, E39D
A02.01
QQSDLQPPA
288
4930.0
70.0
0

MYC, E39D
A02.01
QQQSDLQPPA
287
11835.0
646.0
0

MYC, E39D
A02.01
YQQQQQSDL
289
8842.0
799.0
0.4

MYC, E39D
B08.01
YQQQQQSDL
289
5259.0
18868.0
0

MYC, P57S
A02.01
FELLSTPPL
290
2509.0
225.0
0

MYC, P57S
A02.01
LLSTPPLSPS
291
5226.0
1770.0
0

MYC, P57S
B08.01
FELLSTPPL
290
4208.0
3179.0
0

MYC, T58I
A02.01
LLPIPPLSPS
294
2071.0
449.0
0

MYC, T58I
A02.01
FELLPIPPL
292
2472.0
553.0
0

NAB: STAT6
A02.01
SQIMSLWGL
985
14.0
62.0
1

(“variant 1” of

Chmielecki et al.)

NAB: STAT6
A02.01
IMSLWGLVS
981
3630.0
7321.0
0

(“variant 1” of

Chmielecki et al.)

NAB: STAT6
A24.02
SQIMSLWGL
985
1604.0
8516.0
0

(“variant 1” of

Chmielecki et al.)

NAB: STAT6
B08.01
SQIMSLWGL
985
4587.0
15997.0
0

(“variant 1” of

Chmielecki et al.)

NDRG1: ERG
A02.01
LLQEFDVQEA
988
200.0
45.0
2.5

NDRG1: ERG
A02.01
LQEFDVQEAL
989
2229.0
50280.0
0

NDUFC2
A02.01
ITAFFFCWI
392
437.7
7490.4
not

(-KCDT14)(+1)

measured

NDUFC2
A24.02
LYITAFFFCW
393
46.6
45.3
not

(-KCDT14)(+1)

measured

NDUFC2
A03.01
FFFCWILSCK
391
325.9
597.1
not

(-KCDT14)(+1)

measured

NDUFC2
A03.01
FFCWILSCK
390
985.7
184.9
not

(-KCDT14)(+1)

measured

NDUFC2
A02.01
LLYITAFFL
396
24.0
713.0
17

(-KCDT14)(−1)

NDUFC2
B08.01
LLYITAFFL
396
3588.0
9592.0
0

(-KCDT14)(−1)

NDUFC2
A02.01
ITAFFLLDI
395
699.0
78.7
not

(-KCDT14)(−1)

measured

NDUFC2
A02.01
YITAFFLLDI
400
157.0
64.5
not

(-KCDT14)(−1)

measured

NDUFC2
A24.02
LYITAFFLL
398
15.6
0.1
not

(-KCDT14)(−1)

measured

NDUFC2
A02.01
LLYITAFFLL
397
43.7
323.2
not

(-KCDT14)(−1)

measured

NDUFC2
A24.02
LLYITAFFLL
397
59.7
60.1
not

(-KCDT14)(−1)

measured

NDUFC2
A24.02
LYITAFFLLD
399
414.3
0.4
not

(-KCDT14)(−1)

measured

NRAS, Q61K
A01.01
ILDTAGKEEY
168
272.6
14.3
not

measured

NRAS, Q61R
A01.01
ILDTAGREEY
169
255.8
7.0
not

measured

PDGFRa, T674I
A02.01
IIIEYCFYG
297
693.0
16.0
1.2

PDGFRa, T674I
A02.01
YIIIEYCFYG
300
1529.0
113.0
0

PDGFRa, T674I
A02.01
IIEYCFYGDL
296
3049.0
1090.0
0

PIK3CA, E542K
A02.01
KITEQEKDFL
301
12548.0
1397.0
0

PIK3CA, E542K
A03.01
AISTRDPLSK
1022
41.3
57.5
not

measured

PTEN, R130Q
A02.01
QTGVMICAYL
305
3786.0
9760.0
0

RAC1, P29S
A02.01
FSGEYIPTV
1024
21.0
3.0
6.8

RAC1, P29S
A02.01
AFSGEYIPTV
306
1008.0
781.0
0

RAC1, P29S
A01.01
TTNAFSGEY
1025
23.0
4.4
not

measured

RAC1, P29S
A01.01
YTTNAFSGEY
1026
20.0
10.5
not

measured

RBM27(+1)
B07.02
MPKDVNIQV
402
291.6
12.5
not

measured

RBM27(+1)
A01.01
TGSNEVTTRY
403
343.9
15545.2
not

measured

RBM27(+1)
A01.01
GSNEVTTRY
401
151.6
605.5
not

measured

RNF43, RHTP
A02.01
TQLARFFPI
1111
17.0
19.0
0

(SEQ ID NO:

1140)

RNF43, RHTP
A24.02
TQLARFFPI
1111
268.0
52.0
0

(SEQ ID NO:

1140)

RNF43, RHTP
B08.01
TQLARFFPI
1111
41.0
9150.0
0

(SEQ ID NO:

1140)

SEC31A(−1)
A02.01
KLMLLRLNL
405
58.0
17.0
16.9

SEC31A(−1)
B08.01
KLMLLRLNL
405
421.0
29.0
0

SEC31A(−1)
B07.02
KLMLLRLNL
405
2969.0
133.0
1.5

SEC31A(−1)
A03.01
KLMLLRLNL
405
4664.0
210.0
0

SEC31A(−1)
B08.01
LLRLNLRKM
407
185.1
68.2
not

measured

SEC31A(−1)
A03.01
MLLRLNLRKM
412
171.4
116.9
not

measured

SEC31A(−1)
A03.01
KLMLLRLNLR
406
95.4
48.4
not

measured

SEC31A(−1)
A03.01
MLLRLNLRK
411
14.6
1.3
not

measured

SEC31A(−1)
A03.01
LMLLRLNLRK
409
23.9
6.0
not

measured

SEC31A(−1)
A02.01
MLLRLNLRKM
412
508.5
2507.4
not

measured

SEC31A(−1)
B08.01
MLLRLNLRKM
412
565.9
95.3
not

measured

SEC31A(−1)
A02.01
KLMLLRLNL
405
57.6
2.6
not

measured

SEC31A(−1)
B08.01
LMLLRLNL
408
116.0
9.3
not

measured

SEC31A(−1)
B08.01
KLMLLRLNL
405
420.6
58.4
not

measured

SEC31A(−1)
A02.01
KKLMLLRLNL
404
275.4
288.9
not

measured

SEC31A(−1)
B 08. 01
NLRKMCGPF
413
163.5
35.9
not

measured

SEC31A(−1)
B08.01
YCQKKLMLL
415
203.1
222.1
not

measured

SEC31A(−1)
B 08. 01
LNLRKMCGPF
410
782.2
438.7
not

measured

SEC63 (+1)
A03.01
YTCAITTVK
424
279.0
122.4
not

measured

SEC63 (+1)
A03.01
TYTCAITTVK
423
556.4
2362.2
not

measured

SEC63 (+1)
A03.01
ITTVKATETK
417
795.8
1245.3
not

measured

SEC63 (+1)
A03.01
KSKKKETFKK
419
744.0
39.6
not

measured

SEC63 (+1)
B08.01
TFKKKTYTC
421
648.2
77.9
not

measured

SEC63 (+1)
A03.01
KSKKKETFK
418
411.0
74.3
not

measured

SEC63 (+1)
B08.01
FKKKTYTCAI
416
562.8
384.9
not

measured

SEC63 (−1)
B08.01
TAKSKKRNL
425
213.8
30.6
not

measured

SF3B1, K700E
A02.01
GLVDEQQEV
1027
50.0
44.0
7.4

SLC35F5(−1)
A02.01
FALCGFWQI
426
10.5
0.4
not

measured

SMAP1(−1)
A03.01
KSRQNHLQLK
1112
88.1
4.7
not

measured

SMAP1(−1)
B07.02
KSRQNHLQL
1113
329.5
78.0
not

measured

SMAP1(−1)
A24.02
KLRSPLWIF
1114
504.5
828.2
not

measured

SMAP1(−1)
A03.01
KISNWSLKK
1115
11.5
8.8
not

measured

SMAP1(−1)
A11.01
KISNWSLKK
1115
15.3
9.8
not

measured

SMAP1(−1)
A11.01
SLKKVPALK
1116
117.6
129.1
not

measured

SMAP1(−1)
B08.01
SLKKVPAL
428
66.8
7.9
not

measured

SMAP1(−1)
A03.01
WSLKKVPALK
1117
148.9
94.9
not

measured

SMAP1(−1)
A03.01
KISNWSLKKV
1118
168.3
114.6
not

measured

SMAP1(−1)
A03.01
RKISNWSLKK
429
20.8
130.6
not

measured

SMAP1(−1)
A03.01
SLKKVPALK
1116
29.6
4.4
not

measured

SMAP1(−1)
B08.01
SQKSRQNHL
1119
305.0
44.6
not

measured

SMAP1(−1)
B07.02
ALKKLRSPL
1120
355.5
223.2
not

measured

SMAP1(−1)
B08.01
ALKKLRSPL
1120
58.9
0.5
not

measured

SMAP1(−1)
B08.01
WSLKKVPAL
1121
110.7
12.5
not

measured

SMAP1(−1)
A03.01
HLQLKSCRRK
1122
216.7
96.9
not

measured

SMAP1(−1)
B08.01
LKKLRSPL
427
139.6
0.6
not

measured

SMAP1(−1)
A03.01
SLKKVPALKK
1123
43.1
9.6
not

measured

SPOP, F133L
A02.01
FVQGKDWGL
1028
121.0
34.0
2.1

SPOP, F133L
B08.01
FVQGKDWGL
1028
1401.0
207.0
0

TFAM(+1)
A03.01
RVNTAWKTK
433
136.4
8.6
not

measured

TFAM(+1)
A03.01
RVNTAWKTKK
434
70.6
2.3
not

measured

TFAM(+1)
B08.01
TKKKRVNTA
435
312.4
159.4
not

measured

TFAM(+1)
A03.01
KRVNTAWKTK
431
304.1
331.6
not

measured

TFAM(+1)
B08.01
WKTKKTSFSL
436
930.6
112.2
not

measured

TFAM(+1)
B08.01
MTKKKRVNTA
432
534.2
186.9
not

measured

TGFBR2(−1)
A02.01
RLSSCVPVA
446
83.0
4.0
18.7

TGFBR2(−1)
A03.01
RLSSCVPVA
446
4264.0
439.0
0

TGFBR2(−1)
A03.01
AMTTSSSQK
438
48.5
8.3
not

measured

TGFBR2(−1)
A03.01
AMTTSSSQKN
439
887.2
2336.5
not

measured

TGFBR2(−1)
B08.01
IMKEKKSL
442
69.8
14.1
not

measured

TGFBR2(−1)
A02.01
KSLVRLSSCV
444
903.1
279.8
not

measured

TGFBR2(−1)
A02.01
SLVRLSSCV
449
177.3
29.9
not

measured

TGFBR2(−1)
A11.01
SAMTTSSSQK
448
36.4
15.8
not

measured

TGFBR2(−1)
B08.01
IMKEKKSLV
443
80.8
16.8
not

measured

TGFBR2(−1)
A11.01
AMTTSSSQK
438
89.9
161.6
not

measured

TGFBR2(−1)
A03.01
SAMTTSSSQK
448
96.7
15.7
not

measured

TGFBR2(−1)
A02.01
RLSSCVPVAL
447
84.5
54.2
not

measured

TGFBR2(−1)
A02.01
VRLSSCVPVA
451
640.6
1206.8
not

measured

TGFBR2(−1)
A02.01
RLSSCVPVA
446
82.7
49.5
not

measured

TGFBR2(−1)
B08.01
CIMKEKKSL
440
218.5
7.5
not

measured

TGFBR2(−1)
A02.01
ALMSAMTTS
437
320.4
139.1
not

measured

TGFBR2(−1)
A02.01
LVRLSSCVPV
445
132.7
1237.6
not

measured

THAP5(−1)
A03.01
KMRKKYAQK
452
23.7
5.7
not

measured

TMPRSS2: ERG
A02.01
ALNSEALSV
992
66.0
14.0
9.1

TMPRSS2: ERG
A02.01
ALNSEALSVV
993
84.0
15.0
2.9

TMPRSS2: ERG
A02.01
MALNSEALSV
994
198.0
129.0
0.7

TMPRSS2: ERG
B08.01
MALNSEALSV
994
8512.0
13457.0
0

TP53, AAVG
A02.01
GLLAFWDSQV
815
57.0
10.0
14.2

(SEQ ID NO:

1141)

TP53, AAVG
A02.01
LLAFWDSQV
817
13.0
68.0
12.8

(SEQ ID NO:

1141)

TP53, AWAA
A02.01
WMTETLFDI
849
7.0
14.0
4

(SEQ ID NO:

1142)

TP53, AWAA
A02.01
WMTETLFDIV
850
15.0
40.0
0.4

(SEQ ID NO:

1142)

TP53, AWAA
A24.02
WMTETLFDI
849
4936.0
713.0
0

(SEQ ID NO:

1142)

TP53, AWAA
A01.01
WMTETLFDIV
850
4046.0
14394.0
0

(SEQ ID NO:

1142)

TP53, CSES
B07.02
LPSQRRNHWM
858
89.0
10.0
6.5

(SEQ ID NO:

1143)

TP53, CSES
B08.01
LPSQRRNHWM
858
325.0
47.0
0.7

(SEQ ID NO:

1143)

TP53, CSES
A02.01
ALSEHCPTT
853
208.0
79.0
27.3

(SEQ ID NO:

1143)

TP53, G245S
B08.01
CMGSMNRRPI
1030
1204.0
80.0
0

TP53, G245S
A02.01
YMCNSSCMGS
308
2485.0
81.0
0.8

TP53, G245S
B08.01
SMNRRPILTI
1034
260.0
337.0
0

TP53, G245S
A02.01
SMNRRPILTI
1034
1644.0
1198.0
0.3

TP53, G245S
B08.01
SMNRRPILT
307
2536.0
1282.0
0

TP53, G245S
A02.01
CMGSMNRRPI
1030
7822.0
1989.0
0

TP53, G245S
A02.01
SMNRRPILT
307
7251.0
3839.0
0

TP53, G245S
A24.02
SMNRRPILTI
1034
10308.0
16292.0
0

TP53, G245S
B08.01
GSMNRRPIL
1031
636.7
15.5
not

measured

TP53, G245S
B08.01
MGSMNRRPIL
1033
89.1
6.3
not

measured

TP53, G245S
B08.01
MGSMNRRPI
1032
324.2
29.1
not

measured

TP53, QPSL
B07.02
LPRKPTRAAT
1124
47.0
3.0
3.7

(SEQ ID NO:

1144)

TP53, QPSL
B07.02
LPRKPTRAA
1125
8.0
8.0
5.5

(SEQ ID NO:

1144)

TP53, QPSL
B07.02
KPTRAATVSV
1126
12.0
8.0
3

(SEQ ID NO:

1144)

TP53, QPSL
B08.01
LPRKPTRAA
1125
873.0
1158.0
0

(SEQ ID NO:

1144)

TP53, R248Q
B08.01
NQRPILTII
1037
3433.0
20.0
0

TP53, R248Q
A02.01
GMNQRPILTI
1036
1787.0
709.0
0.4

TP53, R248Q
A02.01
GMNQRPILT
309
8115.0
3029.0
0

TP53, R248Q
A02.01
CMGGMNQRPI
1035
3025.0
3673.0
0

TP53, R248Q
A02.01
NQRPILTII
1037
10855.0
9606.0
0

TP53, R248Q
B08.01
CMGGMNQRPI
1035
6364.0
18766.0
0

TP53, R248Q
B08.01
GMNQRPILTI
1036
3266.0
29251.0
0

TP53, R248W
B08.01
MNWRPILTI
1040
6447.0
1.0
0

TP53, R248W
A02.01
GMNWRPILTI
1039
189.0
282.0
0.5

TP53, R248W
A02.01
CMGGMNWRPI
1038
354.0
346.0
0.4

TP53, R248W
A02.01
MNWRPILTI
1040
5834.0
516.0
3.8

TP53, R248W
A02.01
MNWRPILTII
1041
8158.0
1026.0
0.4

TP53, R248W
B08.01
GMNWRPILTI
1039
3990.0
1045.0
0

TP53, R248W
A02.01
GMNWRPILT
310
3416.0
1130.0
0

TP53, R248W
B08.01
CMGGMNWRPI
1038
3218.0
2248.0
0

TP53, R248W
A24.02
CMGGMNWRPI
1038
9521.0
4453.0
0

TP53, R248W
A24.02
MNWRPILTI
1040
3634.0
6977.0
0.2

TP53, R248W
A24.02
MNWRPILTII
1041
1517.0
44901.0
0

TP53, R273C
A02.01
LLGRNSFEVC
311
1272.0
2081.0
0

TP53, R273C
A02.01
NSFEVCVCA
1042
4239.0
2200.0
0

TP53, R273H
A02.01
NSFEVHVCA
1043
6768.0
503.0
0

TP53, SHST
B07.02
HPRPAPASA
882
13.0
11.0
4.9

(SEQ ID NO:

1145)

TP53, SHST
B08.01
HPRPAPASA
882
1718.0
25.0
0

(SEQ ID NO:

1145)

TP53, Y220C
A02.01
VVPCEPPEV
1044
1268.0
187.0
0.9

TTK(−1)
A02.01
VMSDTTYKI
458
15.8
19.6
not

measured

TTK(−1)
A03.01
LFVMSDTTYK
456
57.9
749.4
not

measured

TTK(−1)
A02.01
FVMSDTTYKI
454
16.0
62.4
not

measured

TTK(−1)
A03.01
FVMSDTTYK
453
63.1
66.9
not

measured

TTK(−1)
A03.01
KTFEKKGEK
455
81.3
32.2
not

measured

TTK(−1)
A01.01
VMSDTTYKIY
459
245.1
375.8
not

measured

TTK(−1)
A01.01
MSDTTYKIY
457
18.9
10.2
not

measured

UBR5(−1)
B07.02
RVQNQGHLL
1127
429.1
826.5
not

measured

VHL, QCIL
A02.01
MLTDSLFLPI
1128
8.0
16.0
1

(SEQ ID NO:

1146)

VHL, QCIL
A02.01
SMLTDSLFL
1129
14.0
31.0
9.8

(SEQ ID NO:

1146)

VHL, QCIL
B08.01
MLTDSLFLPI
1128
2581.0
110.0
0

(SEQ ID NO:

1146)

VHL, QCIL
A01.01
MLTDSLFLPI
1128
429.0
7673.0
0

(SEQ ID NO:

1146)

XPOT(−1)
A02.01
YLTKWPKFFL
460
10.7
42.9
not

measured

TABLE 4A

SEQ ID
Peptide
Measured
Measured

Gene
HLA Allele
Peptide Sequence
NO:
Length
Affinity (nM)
stability (hr.)

KRAS, G12C
A02.01
LVVVGACGV
155
9
667.1
0.6

KRAS, G12C
A02.01
KLVVVGACGV
154
10
70.3
1.0

KRAS, G12D
A02.01
LVVVGADGV
161
9
977.4
0.0

KRAS, G12D
A02.01
KLVVVGADGV
160
10
137.7
0.9

KRAS, G12V
A02.01
LVVVGAVGV
163
9
682.5
0.6

KRAS, G12V
A02.01
KLVVVGAVGV
162
10
57.6
0.9

KRAS, G12C
A03.01
VVGACGVGK
156
9
4.1
5.0

KRAS, G12C
A03.01
VVVGACGVGK
157
10
1.6
2.5

KRAS, G12D
A03.01
VVGADGVGK
158
9
518.7
NB

KRAS, G12D
A03.01
VVVGADGVGK
159
10
314.9
2.3

KRAS, G12V
A03.01
VVGAVGVGK
164
9
1.9
1.2

KRAS, G12V
A03.01
VVVGAVGVGK
5
10
44.2
6.7

KRAS, G12C
A11.01
VVGACGVGK
156
9
43.2
10.0

KRAS, G12C
A11.01
VVVGACGVGK
157
10
69.3
15.7

KRAS, G12D
A11.01
VVGADGVGK
158
9
203.9
3.4

KRAS, G12D
A11.01
VVVGADGVGK
159
10
33.1
13.0

KRAS, G12V
A11.01
VVGAVGVGK
164
9
7.7
16.9

KRAS, G12V
A11.01
VVVGAVGVGK
5
10
26.1
24.3

KRAS, G12D
B08: 01

DGVGKSAL
1147
8

KRAS, G12V
B08: 01

VGVGKSAL
1148
8

KRAS, G12C
B08: 01

CGVGKSAL
1149
8

Table 4B-4M show peptide sequences comprising RAS mutations, corresponding HLA allele to which it binds, and corresponding predicted binding affinity score with the lowest number (e.g., 1) having the highest affinity and vice-versa.

TABLE 4B

RAS Q61H Mutation

SEQ

ID

Rank of

Peptide
NO:
Allele
Binding Potential

ILDTAGHEEY
165
HLA-A36: 01
1

ILDTAGHEEY
165
HLA-A01: 01
2

DTAGHEEYSAM
1150
HLA-A26: 01
3

DTAGHEEYSAM
1150
HLA-A25: 01
4

GHEEYSAM
1151
HLA-B15: 09
4

DTAGHEEY
1152
HLA-A26: 01
5

ILDTAGHEE
1153
HLA-C08: 02
5

AGHEEYSAM
1154
HLA-C01: 02
6

AGHEEYSAM
1154
HLA-B46: 01
6

DTAGHEEY
1152
HLA-A25: 01
6

DTAGHEEY
1152
HLA-A01: 01
6

DTAGHEEY
1152
HLA-B18: 01
7

DTAGHEEY
1152
HLA-A36: 01
7

ILDTAGHEE
1153
HLA-C05: 01
7

ILDTAGHEE
1153
HLA-A02: 07
7

ILDTAGHEEY
165
HLA-A29: 02
7

ILDTAGHEEY
165
HLA-C08: 02
7

HEEYSAMRD
1155
HLA-B49: 01
8

TAGHEEYSA
1156
HLA-B35: 03
8

DTAGHEEYS
1157
HLA-A68: 02
9

DTAGHEEYSAMR
1158
HLA-A68: 01
9

GHEEYSAM
1151
HLA-B39: 01
9

ILDTAGHEE
1153
HLA-A01: 01
9

LDTAGHEEY
1159
HLA-B53: 01
9

HEEYSAMRD
1155
HLA-B41: 01
10

ILDTAGHEE
1153
HLA-A36: 01
10

DTAGHEEY
1152
HLA-B58: 01
11

LLDILDTAGH
1160
HLA-A01: 01
12

TAGHEEYSAM
1161
HLA-B35: 03
12

LDTAGHEEY
1159
HLA-B35: 01
13

DILDTAGHE
1162
HLA-A26: 01
14

DTAGHEEY
1152
HLA-C12: 03
14

ILDTAGHEEY
165
HLA-C05: 01
14

AGHEEYSAM
1154
HLA-A30: 02
15

DILDTAGHEEY
1163
HLA-A25: 01
15

DTAGHEEY
1152
HLA-C02: 02
15

ILDTAGHEE
1153
HLA-C04: 01
15

DILDTAGH
1164
HLA-A26: 01
16

ILDTAGHEE
1153
HLA-A02: 01
16

LDTAGHEEY
1159
HLA-A29: 02
16

ILDTAGHE
1165
HLA-A01: 01
17

LDTAGHEEY
1159
HLA-B18: 01
17

AGHEEYSAM
1154
HLA-C14: 03
18

DILDTAGHEEY
1163
HLA-A29: 02
18

DTAGHEEYS
1157
HLA-A26: 01
18

ILDTAGHEEY
165
HLA-B15: 01
18

DTAGHEEYSA
1166
HLA-A68: 02
19

ILDTAGHE
1165
HLA-C05: 01
19

ILDTAGHEEY
165
HLA-A02: 07
19

ILDTAGHEEY
165
HLA-A30: 02
19

LDTAGHEEY
1159
HLA-A36: 01
19

AGHEEYSAM
1154
HLA-C14: 02
20

AGHEEYSAM
1154
HLA-B15: 03
20

LLDILDTAGH
1160
HLA-A02: 07
20

TABLE 4C

RAS Q61R Mutation

SEQ

ID

Rank of

Peptide
NO:
Allele
Binding Potential

ILDTAGREEY
169
HLA-A36: 01
1

ILDTAGREEY
169
HLA-A01: 01
2

DTAGREEYSAM
1167
HLA-A26: 01
3

DILDTAGR
1168
HLA-A33: 03
4

DILDTAGR
1168
HLA-A68: 01
5

DTAGREEY
1169
HLA-A26: 01
6

DTAGREEYSAM
1167
HLA-A25: 01
6

CLLDILDTAGR
1170
HLA-A74: 01
7

DTAGREEY
1169
HLA-A01: 01
7

REEYSAMRD
1171
HLA-B41: 01
7

GREEYSAMR
1172
HLA-B27: 05
8

ILDTAGREE
1173
HLA-C08: 02
8

ILDTAGREEY
169
HLA-A29: 02
8

REEYSAMRD
1171
HLA-B49: 01
8

AGREEYSAM
1174
HLA-B46: 01
9

DTAGREEY
1169
HLA-B18: 01
9

DTAGREEY
1169
HLA-A25: 01
9

DTAGREEY
1169
HLA-A36: 01
9

DILDTAGR
1168
HLA-A74: 01
10

DILDTAGRE
1175
HLA-A26: 01
10

ILDTAGREE
1173
HLA-C05: 01
10

DILDTAGR
1168
HLA-A26: 01
11

GREEYSAM
1176
HLA-B39: 01
11

AGREEYSAM
1174
HLA-B15: 03
12

GREEYSAM
1176
HLA-C07: 02
12

ILDTAGREE
1173
HLA-A01: 01
12

TAGREEYSA
1177
HLA-B35: 03
12

ILDTAGREEY
169
HLA-A30: 02
13

DTAGREEYS
1178
HLA-A68: 02
14

ILDTAGRE
1179
HLA-A01: 01
14

CLLDILDTAGR
1170
HLA-A31: 01
15

DTAGREEYSAMR
1180
HLA-A68: 01
15

LLDILDTAGR
1181
HLA-A01: 01
15

DTAGREEY
1169
HLA-B58: 01
16

ILDTAGREEY
169
HLA-C08: 02
16

DILDTAGR
1168
HLA-A31: 01
17

ILDTAGREE
1173
HLA-C04: 01
17

ILDTAGREEY
169
HLA-A32: 01
17

LLDILDTAGR
1181
HLA-A74: 01
17

TAGREEYSAM
1182
HLA-B35: 03
17

DILDTAGREEY
1183
HLA-A32: 01
18

ILDTAGRE
1179
HLA-C05: 01
18

ILDTAGREE
1173
HLA-A02: 07
18

REEYSAMRD
1171
HLA-B40: 01
18

AGREEYSAM
1174
HLA-B15: 01
19

AGREEYSAMR
1184
HLA-A31: 01
19

ILDTAGRE
1179
HLA-A36: 01
19

LDILDTAGR
1185
HLA-A68: 01
19

LDTAGREEY
1186
HLA-A29: 02
19

LDTAGREEY
1186
HLA-B35: 01
19

REEYSAMRD
1171
HLA-B45: 01
19

REEYSAMRDQY
1187
HLA-A36: 01
19

DTAGREEY
1169
HLA-C02: 02
20

TABLE 4D

RAS Q61K Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

ILDTAGKEEY
168
HLA-A36:01
1

ILDTAGKEEY
168
HLA-A01:01
2

DTAGKEEYSAM
1188
HLA-A26:01
3

CLLDILDTAGK
1189
HLA-A03:01
4

DTAGKEEY
1190
HLA-A01:01
5

DTAGKEEY
1190
HLA-A26:01
5

DTAGKEEYSAM
1188
HLA-A25:01
5

AGKEEYSAM
1191
HLA-B46:01
6

DILDTAGKE
1192
HLA-A26:01
7

KEEYSAMRD
1193
HLA-B41:01
7

DTAGKEEY
1190
HLA-B18:01
8

GKEEYSAM
1194
HLA-B15:03
8

ILDTAGKEE
1195
HLA-C08:02
8

ILDTAGKEEY
168
HLA-A29:02
8

DTAGKEEYS
1196
HLA-A68:02
9

LDTAGKEEY
1197
HLA-B53:01
9

TAGKEEYSA
1198
HLA-B35:03
9

DILDTAGK
1199
HLA-A68:01
10

DTAGKEEY
1190
HLA-A36:01
10

KEEYSAMRD
1193
HLA-B49:01
10

LDTAGKEEY
1197
HLA-C07:01
10

DTAGKEEYSAMR
1200
HLA-A68:01
11

ILDTAGKEE
1195
HLA-C05:01
11

ILDTAGKEEY
168
HLA-C08:02
11

LLDILDTAGK
1201
HLA-A01:01
12

AGKEEYSAM
1191
HLA-A30:02
13

DTAGKEEY
1190
HLA-A25:01
13

DTAGKEEYS
1196
HLA-A26:01
13

ILDTAGKE
1202
HLA-C05:01
13

LDTAGKEEY
1197
HLA-B35:01
13

AGKEEYSAMR
1203
HLA-A31:01
14

DILDTAGK
1199
HLA-A33:03
14

ILDTAGKE
1202
HLA-A01:01
14

ILDTAGKEE
1195
HLA-A01:01
14

ILDTAGKEE
1195
HLA-A02:07
14

TAGKEEYSAM
1204
HLA-B35:03
14

AGKEEYSAM
1191
HLA-B15:01
15

ILDTAGKEEY
168
HLA-A30:02
15

LDTAGKEEY
1197
HLA-B46:01
15

DTAGKEEY
1190
HLA-B58:01
16

ILDTAGKEEY
168
HLA-C05:01
17

AGKEEYSAM
1191
HLA-A30:01
18

AGKEEYSAM
1191
HLA-B15:03
18

DTAGKEEY
1190
HLA-C02:02
18

LDTAGKEEY
1197
HLA-A29:02
18

TABLE 4E

RAS Q61L Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

ILDTAGLEEY
166
HLA-A36:01
1

ILDTAGLEEY
166
HLA-A01:01
2

LLDILDTAGL
167
HLA-A02:07
3

GLEEYSAMRDQY
1205
HLA-A36:01
4

DTAGLEEY
1206
HLA-A25:01
5

DTAGLEEY
1206
HLA-A26:01
5

DTAGLEEYSAM
1207
HLA-A26:01
5

DTAGLEEY
1206
HLA-A01:01
6

ILDTAGLEE
1208
HLA-C08:02
6

ILDTAGLEE
1208
HLA-A01:01
6

CLLDILDTAGL
1209
HLA-A02:04
7

ILDTAGLEE
1208
HLA-A36:01
7

LLDILDTAGL
167
HLA-A01:01
7

DILDTAGL
1210
HLA-B14:02
8

DILDTAGLEEY
1211
HLA-A25:01
8

DTAGLEEYS
1212
HLA-A68:02
8

DTAGLEEYSAM
1207
HLA-A25:01
8

GLEEYSAMR
1213
HLA-A74:01
8

ILDTAGLE
1214
HLA-A01:01
8

DILDTAGLEEY
1211
HLA-A26:01
9

DTAGLEEY
1206
HLA-A36:01
9

ILDTAGLEEY
166
HLA-A29:02
9

DILDTAGL
1210
HLA-B08:01
10

DTAGLEEY
1206
HLA-B18:01
10

ILDTAGLEE
1208
HLA-A02:07
10

LDTAGLEEY
1215
HLA-B35:01
10

CLLDILDTAGL
1209
HLA-A02:01
11

DTAGLEEY
1206
HLA-C02:02
11

ILDTAGLEE
1208
HLA-C05:01
11

ILDTAGLEEY
166
HLA-C08:02
11

ILDTAGLEEY
166
HLA-A02:07
11

LLDILDTAGL
167
HLA-C08:02
11

DILDTAGL
1210
HLA-A26:01
12

LDTAGLEEY
1215
HLA-B53:01
12

DTAGLEEY
1206
HLA-C03:02
13

DTAGLEEY
1206
HLA-B58:01
13

ILDTAGLEEY
166
HLA-A30:02
13

LLDILDTAGL
167
HLA-C05:01
13

LLDILDTAGL
167
HLA-C04:01
13

DTAGLEEYSAMR
1216
HLA-A68:01
14

ILDTAGLE
1214
HLA-A36:01
15

LLDILDTAGL
167
HLA-A02:01
15

AGLEEYSAM
1217
HLA-B15:03
16

DTAGLEEYSA
1218
HLA-A68:02
16

GLEEYSAMRDQY
1205
HLA-A01:01
16

ILDTAGLE
1214
HLA-C04:01
16

ILDTAGLEEY
166
HLA-B15:01
16

LDILDTAGL
1219
HLA-B37:01
16

AGLEEYSAM
1217
HLA-A30:02
17

AGLEEYSAM
1217
HLA-B48:01
17

AGLEEYSAMR
1220
HLA-A31:01
17

ILDTAGLEE
1208
HLA-C04:01
17

LDTAGLEEY
1215
HLA-C03:02
17

AGLEEYSAM
1217
HLA-C14:02
18

GLEEYSAMR
1213
HLA-A31:01
18

LEEYSAMRD
1221
HLA-B41:01
18

LLDILDTAGLE
1222
HLA-A01:01
18

AGLEEYSAM
1217
HLA-C14:03
19

LDILDTAGL
1219
HLA-B40:02
19

LDTAGLEEY
1215
HLA-A29:02
19

DILDTAGLE
1223
HLA-A26:01
20

DTAGLEEY
1206
HLA-B15:01
20

ILDTAGLEEY
166
HLA-A02:01
20

LDTAGLEEY
1215
HLA-A36:01
20

LDTAGLEEY
1215
HLA-B46:01
20

DTAGLEEY
1206
HLA-A68:02
21

DTAGLEEY
1206
HLA-C12:03
21

ILDTAGLE
1214
HLA-C05:01
21

LDTAGLEEY
1215
HLA-B18:01
21

LEEYSAMRD
1221
HLA-B49:01
21

TAGLEEYSA
1224
HLA-B54:01
21

DILDTAGLEEY
1211
HLA-A29:02
22

GLEEYSAM
1225
HLA-C05:01
22

TABLE 4F

RAS G12A Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

AAGVGKSAL
1226
HLA-C03:04
1

VVVGAAGVGK
1227
HLA-A11:01
1

VVGAAGVGK
1228
HLA-A11:01
2

TEYKLVVVGAA
1229
HLA-B50:01
3

VVGAAGVGK
1228
HLA-A03:01
3

VVVGAAGVGK
1227
HLA-A68:01
3

AAGVGKSAL
1226
HLA-C08:02
4

AAGVGKSAL
1226
HLA-C08:01
4

AAGVGKSAL
1226
HLA-B46:01
4

AAGVGKSAL
1226
HLA-B81:01
5

GAAGVGKSAL
1230
HLA-B48:01
5

LVVVGAAGV
1231
HLA-A68:02
5

AAGVGKSAL
1226
HLA-C03:04
1

VVVGAAGVGK
1227
HLA-A11:01
1

VVGAAGVGK
1228
HLA-A11:01
2

TEYKLVVVGAA
1229
HLA-B50:01
3

VVGAAGVGK
1228
HLA-A03:01
3

VVVGAAGVGK
1227
HLA-A68:01
3

AAGVGKSAL
1226
HLA-C08:02
4

AAGVGKSAL
1226
HLA-C08:01
4

AAGVGKSAL
1226
HLA-B46:01
4

AAGVGKSAL
1226
HLA-B81:01
5

AAGVGKSAL
1226
HLA-C03:02
5

AAGVGKSAL
1226
HLA-C01:02
5

GAAGVGKSAL
1230
HLA-B48:01
5

LVVVGAAGV
1231
HLA-A68:02
5

AAGVGKSAL
1226
HLA-C03:03
6

VVGAAGVGK
1228
HLA-A68:01
6

GAAGVGKSAL
1230
HLA-B81:01
7

VVVGAAGVGK
1227
HLA-A03:01
7

AAGVGKSAL
1226
HLA-C05:01
8

AAGVGKSAL
1226
HLA-C12:03
8

GAAGVGKSA
1232
HLA-B46:01
8

VVGAAGVGK
1228
HLA-A30:01
8

GAAGVGKSA
1232
HLA-B55:01
9

KLVVVGAAGV
1233
HLA-A02:01
9

AGVGKSAL
1234
HLA-B08:01
10

GAAGVGKSAL
1230
HLA-C03:04
10

AAGVGKSAL
1226
HLA-C17:01
11

GAAGVGKSAL
1230
HLA-C03:03
11

VVVGAAGV
1235
HLA-A68:02
11

YKLVVVGAA
1236
HLA-B54:01
11

AAGVGKSAL
1226
HLA-B48:01
12

AGVGKSAL
1234
HLA-C03:04
12

AGVGKSAL
1234
HLA-C07:01
12

VVVGAAGVGK
1227
HLA-A30:01
12

AAGVGKSA
1237
HLA-B46:01
13

KLVVVGAAGV
1233
HLA-A02:07
13

YKLVVVGAA
1236
HLA-B50:01
13

AAGVGKSAL
1226
HLA-B07:02
14

GAAGVGKSAL
1230
HLA-A68:02
14

VVGAAGVGK
1228
HLA-A74:01
14

AGVGKSAL
1234
HLA-C08:01
15

GAAGVGKSAL
1230
HLA-C17:01
15

GAAGVGKSAL
1230
HLA-C08:01
16

GAAGVGKSAL
1230
HLA-B35:03
16

AAGVGKSAL
1226
HLA-C02:02
17

AAGVGKSAL
1226
HLA-B35:03
17

AAGVGKSAL
1226
HLA-C12:02
17

AAGVGKSAL
1226
HLA-C14:03
17

GAAGVGKSA
1232
HLA-B50:01
17

AGVGKSAL
1234
HLA-C03:02
18

GAAGVGKSA
1232
HLA-C03:04
18

LVVVGAAGV
1231
HLA-B55:01
18

TEYKLVVVGAA
1229
HLA-B41:01
18

AGVGKSAL
1234
HLA-C01:02
19

GAAGVGKSA
1232
HLA-B54:01
19

GAAGVGKSAL
1230
HLA-B07:02
19

VGAAGVGKSA
1238
HLA-B55:01
19

AGVGKSAL
1234
HLA-B48:01
20

AGVGKSALTI
1239
HLA-B49:01
20

VVVGAAGV
1235
HLA-B55:01
20

TABLE 4G

RAS G12C Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

VVVGACGVGK
157
HLA-A11:01
1

VVGACGVGK
156
HLA-A03:01
2

VVGACGVGK
156
HLA-A11:01
3

VVVGACGVGK
157
HLA-A68:01
4

VVGACGVGK
156
HLA-A68:01
5

VVVGACGVGK
157
HLA-A03:01
5

VVGACGVGK
156
HLA-A30:01
6

ACGVGKSAL
1240
HLA-B81:01
7

ACGVGKSAL
1240
HLA-C01:02
7

ACGVGKSAL
1240
HLA-C14:03
8

ACGVGKSAL
1240
HLA-C03:04
9

VVVGACGVGK
157
HLA-A30:01
9

ACGVGKSAL
1240
HLA-C14:02
10

CGVGKSAL
1149
HLA-B08:01
10

KLVVVGACGV
154
HLA-A02:01
10

ACGVGKSAL
1240
HLA-B07:02
11

GACGVGKSAL
1241
HLA-B48:01
12

GACGVGKSAL
1241
HLA-C03:03
13

ACGVGKSAL
1240
HLA-B48:01
14

ACGVGKSAL
1240
HLA-B40:01
14

YKLVVVGAC
1242
HLA-B48:01
14

YKLVVVGAC
1242
HLA-B15:03
14

GACGVGKSA
1243
HLA-B46:01
15

GACGVGKSAL
1241
HLA-C03:04
15

GACGVGKSAL
1241
HLA-C01:02
15

LVVVGACGV
155
HLA-A68:02
15

CGVGKSAL
1149
HLA-C03:04
16

GACGVGKSAL
1241
HLA-C08:02
16

VVGACGVGK
156
HLA-A74:01
16

TABLE 4H

RAS G12D Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

GADGVGKSAL
1244
HLA-C08:02
1

GADGVGKSAL
1244
HLA-C05:01
2

VVVGADGVGK
159
HLA-A11:01
3

DGVGKSAL
1147
HLA-B14:02
4

VVGADGVGK
158
HLA-A11:01
4

VVGADGVGK
158
HLA-A03:01
5

DGVGKSAL
1147
HLA-B08:01
6

VVVGADGVGK
159
HLA-A68:01
6

GADGVGKSAL
1244
HLA-C03:03
7

VVGADGVGK
158
HLA-A30:01
7

ADGVGKSAL
1245
HLA-B37:01
8

GADGVGKSAL
1244
HLA-C08:01
8

VVGADGVGK
158
HLA-A68:01
8

GADGVGKSA
1246
HLA-C08:02
9

GADGVGKSAL
1244
HLA-B35:03
9

GADGVGKS
1247
HLA-C05:01
10

GADGVGKSA
1246
HLA-C05:01
10

ADGVGKSAL
1245
HLA-C07:01
11

VVVGADGVGK
159
HLA-A03:01
11

ADGVGKSAL
1245
HLA-B40:02
12

ADGVGKSAL
1245
HLA-B46:01
13

GADGVGKSAL
1244
HLA-C03:04
13

ADGVGKSAL
1245
HLA-B81:01
14

GADGVGKSAL
1244
HLA-C17:01
14

VVVGADGVGK
159
HLA-A30:01
14

GADGVGKSA
1246
HLA-B35:03
15

GADGVGKSA
1246
HLA-B46:01
15

GADGVGKSAL
1244
HLA-B48:01
15

KLVVVGADGV
160
HLA-A02:01
15

LVVVGADGV
161
HLA-A68:02
15

VGADGVGKSA
1248
HLA-B55:01
15

VVGADGVGK
158
HLA-A74:01
16

GADGVGKSA
1246
HLA-B53:01
17

KLVVVGADGV
160
HLA-A02:07
17

VGADGVGK
1249
HLA-A68:01
17

YKLVVVGAD
1250
HLA-B48:01
17

ADGVGKSAL
1245
HLA-C14:03
18

DGVGKSALTI
1251
HLA-B51:01
18

VGADGVGK
1249
HLA-A11:01
18

GADGVGKSAL
1244
HLA-B07:02
19

KLVVVGADGVGK
1252
HLA-A03:01
20

TABLE 4I

RAS G12R Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

VVGARGVGK
1
HLA-A03:01
1

EYKLVVVGAR
2
HLA-A33:03
2

VVVGARGVGK
3
HLA-A11:01
3

ARGVGKSAL
4
HLA-C07:02
4

ARGVGKSAL
4
HLA-B39:01
5

ARGVGKSAL
4
HLA-C07:01
5

VVGARGVGK
1
HLA-A11:01
5

VVVGARGVGK
3
HLA-A68:01
5

GARGVGKSA
1253
HLA-B46:01
6

ARGVGKSAL
4
HLA-B27:05
7

GARGVGKSA
1253
HLA-B55:01
7

RGVGKSAL
1254
HLA-C07:01
8

VVGARGVGK
1
HLA-A30:01
9

ARGVGKSAL
4
HLA-B38:01
10

ARGVGKSAL
4
HLA-B14:02
10

VVGARGVGK
1
HLA-A68:01
10

VVVGARGVGK
3
HLA-A03:01
11

GARGVGKSAL
1255
HLA-B48:01
12

RGVGKSAL
1254
HLA-B48:01
12

RGVGKSALTI
1256
HLA-A23:01
12

ARGVGKSAL
4
HLA-C06:02
13

GARGVGKSA
1253
HLA-A30:01
13

GARGVGKSAL
1255
HLA-B81:01
13

VVVGARGVGK
3
HLA-A30:01
13

GARGVGKSAL
1255
HLA-B07:02
14

LVVVGARGV
1257
HLA-C06:02
14

RGVGKSAL
1254
HLA-B81:01
14

VVGARGVGK
1
HLA-A74:01
15

KLVVVGARGV
1258
HLA-A02:01
16

LVVVGARGV
1257
HLA-B55:01
16

YKLVVVGAR
1259
HLA-A33:03
16

KLVVVGAR
1260
HLA-A74:01
17

KLVVVGARGV
1258
HLA-B13:02
17

RGVGKSAL
1254
HLA-C01:02
17

LVVVGARGV
1257
HLA-A68:02
18

VVVGARGV
1261
HLA-B55:01
18

ARGVGKSAL
4
HLA-B15:09
19

ARGVGKSAL
4
HLA-C14:03
20

GARGVGKSA
1253
HLA-B54:01
20

VVVGARGV
1261
HLA-B52:01
20

KLVVVGARGVGK
1262
HLA-A03:01
21

TABLE 4J

RAS G12S Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

VVVGASGVGK
1263
HLA-A11:01
1

VVGASGVGK
1264
HLA-A11:01
2

VVGASGVGK
1264
HLA-A03:01
3

VVVGASGVGK
1263
HLA-A68:01
4

ASGVGKSAL
1265
HLA-C03:04
5

ASGVGKSAL
1265
HLA-B46:01
5

VVGASGVGK
1264
HLA-A68:01
6

VVVGASGVGK
1263
HLA-A03:01
6

ASGVGKSAL
1265
HLA-C01:02
7

GASGVGKSAL
1266
HLA-B48:01
7

ASGVGKSAL
1265
HLA-C07:01
8

ASGVGKSAL
1265
HLA-C08:02
9

GASGVGKSAL
1266
HLA-B81:01
9

SGVGKSAL
1267
HLA-B08:01
9

ASGVGKSAL
1265
HLA-C03:03
10

ASGVGKSAL
1265
HLA-C03:02
10

SGVGKSAL
1267
HLA-B14:02
10

VVGASGVGK
1264
HLA-A30:01
10

ASGVGKSAL
1265
HLA-C08:01
11

VVVGASGVGK
1263
HLA-A30:01
11

GASGVGKSAL
1266
HLA-B35:03
12

SGVGKSAL
1267
HLA-C07:01
12

ASGVGKSAL
1265
HLA-B81:01
13

GASGVGKSA
1268
HLA-B55:01
13

GASGVGKSAL
1266
HLA-C03:03
13

KLVVVGASGV
1269
HLA-A02:01
13

LVVVGASGV
1270
HLA-A68:02
13

SGVGKSAL
1267
HLA-C01:02
13

ASGVGKSA
1271
HLA-B46:01
14

ASGVGKSAL
1265
HLA-C15:02
14

GASGVGKSAL
1266
HLA-C08:01
15

SGVGKSAL
1267
HLA-C03:04
15

ASGVGKSAL
1265
HLA-C05:01
16

GASGVGKSAL
1266
HLA-C03:04
16

VVGASGVGK
1264
HLA-A74:01
16

ASGVGKSAL
1265
HLA-B48:01
17

GASGVGKSAL
1266
HLA-C01:02
17

SGVGKSAL
1267
HLA-C03:02
17

SGVGKSALTI
1272
HLA-A23:01
17

VGASGVGKSA
1273
HLA-B55:01
18

ASGVGKSAL
1265
HLA-C12:03
19

ASGVGKSAL
1265
HLA-B57:03
19

KLVVVGASGV
1269
HLA-A02:07
19

SGVGKSAL
1267
HLA-B81:01
19

ASGVGKSAL
1265
HLA-C17:01
20

KLVVVGASG
1274
HLA-A32:01
20

TABLE 4K

RAS G12V Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

VVGAVGVGK
164
HLA-A03:01
1

VVGAVGVGK
164
HLA-A11:01
2

VVVGAVGVGK
5
HLA-A11:01
2

VVVGAVGVGK
5
HLA-A68:01
3

VVGAVGVGK
164
HLA-A68:01
4

LVVVGAVGV
163
HLA-A68:02
5

VVGAVGVGK
164
HLA-A30:01
5

AVGVGKSAL
1275
HLA-B81:01
6

KLVVVGAVGV
162
HLA-A02:01
6

AVGVGKSAL
1275
HLA-B46:01
7

GAVGVGKSAL
1276
HLA-C03:03
7

GAVGVGKSAL
1276
HLA-B48:01
7

VVVGAVGVGK
5
HLA-A03:01
7

AVGVGKSAL
1275
HLA-C03:04
8

GAVGVGKSAL
1276
HLA-C03:04
8

KLVVVGAVGV
162
HLA-A02:07
9

VGVGKSAL
1148
HLA-B08:01
9

VVVGAVGV
1277
HLA-A68:02
9

AVGVGKSAL
1275
HLA-C08:02
10

AVGVGKSAL
1275
HLA-B07:02
10

GAVGVGKSAL
1276
HLA-B35:03
10

AVGVGKSAL
1275
HLA-C08:01
11

AVGVGKSAL
1275
HLA-C01:02
11

GAVGVGKSA
1278
HLA-B55:01
11

GAVGVGKSAL
1276
HLA-B81:01
11

GAVGVGKSAL
1276
HLA-C08:01
11

KLVVVGAVGV
162
HLA-B13:02
11

VGVGKSAL
1148
HLA-C03:04
11

AVGVGKSAL
1275
HLA-A32:01
12

GAVGVGKSA
1278
HLA-B46:01
12

VGVGKSAL
1148
HLA-C03:02
12

VGVGKSALTI
1279
HLA-A23:01
12

GAVGVGKSA
1278
HLA-B54:01
13

VGVGKSAL
1148
HLA-C01:02
.3

AVGVGKSAL
1275
HLA-B48:01
14

AVGVGKSAL
1275
HLA-C03:03
14

AVGVGKSAL
1275
HLA-B42:01
14

LVVVGAVGV
163
HLA-B55:01
14

VGVGKSAL
1148
HLA-C08:01
14

VVGAVGVGK
164
HLA-A74:01
14

AVGVGKSAL
1275
HLA-C05:01
15

AVGVGKSAL
1275
HLA-C03:02
15

GAVGVGKSA
1278
HLA-C03:04
15

KLVVVGAVGV
162
HLA-A02:04
15

LVVVGAVGV
163
HLA-A02:07
15

VGVGKSAL
1148
HLA-B14:02
15

VVVGAVGVGK
5
HLA-A30:01
15

VVGAVGVGK
164
HLA-B81:01
16

VVVGAVGV
1277
HLA-B55:01
16

AVGVGKSAL
1275
HLA-C14:03
17

AVGVGKSAL
1275
HLA-B15:01
17

LVVVGAVGV
163
HLA-B54:01
17

AVGVGKSA
1280
HLA-B55:01
18

AVGVGKSAL
1275
HLA-C17:01
18

GAVGVGKSA
1278
HLA-B50:01
19

GAVGVGKSAL
1276
HLA-C17:01
19

YKLVVVGAV
1281
HLA-A02:04
19

GAVGVGKSAL
1276
HLA-B35:01
20

VVGAVGVGK
164
HLA-A31:01
20

YKLVVVGAV
1281
HLA-B51:01
20

LVVVGAVGVGK
1282
HLA-A03:01
21

KLVVVGAVGVGK
1283
HLA-A03:01
22

TABLE 4L

RAS G13C Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

VVVGAGCVGK
1284
HLA-A11:01
1

VVGAGCVGK
1285
HLA-A11:01
2

AGCVGKSAL
1286
HLA-C01:02
3

VVGAGCVGK
1285
HLA-A03:01
4

VVVGAGCVGK
1284
HLA-A68:01
4

CVGKSALTI
1287
HLA-B13:02
5

VVGAGCVGK
1285
HLA-A68:01
5

VVGAGCVGK
1285
HLA-A30:01
6

AGCVGKSAL
1286
HLA-B48:01
7

AGCVGKSAL
1286
HLA-C03:04
8

GCVGKSALTI
1288
HLA-B49:01
8

AGCVGKSAL
1286
HLA-C08:02
9

VVVGAGCVGK
1284
HLA-A03:01
9

KLVVVGAGC
1289
HLA-A30:02
10

GCVGKSAL
1290
HLA-C07:01
11

VVGAGCVGK
1285
HLA-A74:01
12

AGCVGKSAL
1286
HLA-C14:03
13

KLVVVGAGC
1289
HLA-B15:01
14

TABLE 4M

RAS G13D Mutation

Rank of

Binding

Peptide
SEQ ID NO:
Allele
Potential

AGDVGKSAL
1291
HLA-C08:02
1

AGDVGKSAL
1291
HLA-C05:01
2

VVGAGDVGK
1292
HLA-A1L01
3

VVVGAGDVGK
1293
HLA-A1L01
3

VVVGAGDVGK
1293
HLA-A68:01
4

GAGDVGKSA
1294
HLA-B46:01
5

GAGDVGKSAL
1295
HLA-B48:01
5

VVGAGDVGK
1292
HLA-A68:01
5

VVGAGDVGK
1292
HLA-A03:01
5

AGDVGKSAL
1291
HLA-C03:04
6

AGDVGKSAL
1291
HLA-C04:01
6

AGDVGKSAL
1291
HLA-C0L02
6

DVGKSALTI
1296
HLA-B13:02
6

DVGKSALTI
1296
HLA-A25:01
6

GDVGKSAL
1297
HLA-C07:01
6

GDVGKSAL
1297
HLA-B40:02
7

GDVGKSAL
1297
HLA-B37:01
8

AGDVGKSAL
1291
HLA-B48:01
9

DVGKSALTI
1296
HLA-B51:01
10

VVGAGDVGK
1292
HLA-A30:01
10

GAGDVGKSAL
1295
HLA-C08:01
11

GAGDVGKSAL
1295
HLA-B81:01
11

AGDVGKSAL
1291
HLA-C08:01
12

GAGDVGKSAL
1295
HLA-C03:04
12

DVGKSALTI
1296
HLA-B53:01
13

AGDVGKSAL
1291
HLA-B07:02
14

AGDVGKSAL
1291
HLA-B46:01
14

DVGKSALTI
1296
HLA-A26:01
14

VVGAGDVGK
1292
HLA-A74:01
14

GAGDVGKSA
1294
HLA-B54:01
15

DVGKSALTI
1296
HLA-B38:01
16

GAGDVGKSAL
1295
HLA-C03:03
16

VVVGAGDVGK
1293
HLA-A03:01
16

Table 5 shows GATA peptides and their HLA binding partners.

TABLE 5

Exemplary

Protein
Mutation Sequence
Peptides (HLA allele
Exemplary

Gene
Change
Context
example(s))
Diseases

FRAMESHIFT¹

GATA3
L328fs

AQAKAVCSQESRDV

CLQCLWALL (SEQ ID NO:
Breast Cancer

N334fs

LCELSDHHNHTLEEE

1299)(A02.01)

CQWGPCLQCLWALL

CQWGPCLQCL (SEQ ID NO:

QASQY* (SEQ ID NO:
1300)(A02.01)

1298)
QWGPCLQCL (SEQ ID NO:

1301)(A24.02)

QWGPCLQCLW (SEQ ID NO:

1302)(A24.02)

GATA3
H400fs

PGRPLQTHVLPEPHL

AIQPVLWTT (SEQ ID NO:
Breast Cancer

S408fs

ALQPLQPHADHAHA

1303)(A02.01)

S408fs

DAPAIQPVLWTTPPL

ALQPLQPHA (SEQ ID NO:

S430fs

QHGHRHGLEPCSML

1304)(A02.01)

H434fs

TGPPARVPAVPFDLH

DLHFCRSSIM (SEQ ID NO:

H435fs

FCRSSIMKPKRDGYM

1305)(B08.01)

FLKAESKIMFATLQR

EPHLALQPL (SEQ ID NO:

SSLWCLCSNH* (SEQ
1306)(B07.02, B08.01)

ID NO: 111)
ESKIMFATL (SEQ ID NO: 1307)

(B08.01)

FATLQRSSL (SEQ ID NO: 1308)

(B07.02, B08.01)

FLKAESKIM (SEQ ID NO:

1309)(B08.01)

FLKAESKIMF (SEQ ID NO:

1310)(B08.01)

GPPARVPAV (SEQ ID NO:

1311)(B07.02)

IMKPKRDGYM (SEQ ID NO:

1312)(B08.01)

KIMFATLQR (SEQ ID NO:

1313)(A03.01)

KPKRDGYMF (SEQ ID NO:

1314)(B07.02)

KPKRDGYMFL (SEQ ID NO:

1315)(B07.02)

LHFCRSSIM (SEQ ID NO: 1316)

(B08.01)

LQHGHRHGL (SEQ ID NO:

1317)(B08.01)

MFATLQRSSL (SEQ ID NO:

1318)(B07.02, B08.01)

MFLKAESKI (SEQ ID NO:

1319)(A24.02)

MLTGPPARV (SEQ ID NO:

1320)(A02.01)

QPVLWTTPPL (SEQ ID NO:

1321)(B07.02)

SMLTGPPARV (SEQ ID NO:

1322)(A02.01)

TLQRSSLWCL (SEQ ID NO:

1323)(A02.01)

VLPEPHLAL (SEQ ID NO:

1324)(A02.01)

VPAVPFDLHF (SEQ ID NO:

1325)(B07.02)

YMFLKAESK (SEQ ID NO:

1326)(A03.01)

YMFLKAESKI (SEQ ID NO:

1327)(A02.01, A03.01, A24.02,

B08.01)

Table 6 shows HLA affinity and stability of selected BTK peptides:

TABLE 6

SEQ

Stability

HLA
Peptide
ID
Affinity
(half-life

Gene
Allele
Sequence
NO:
(nM)
(hr.))

BTK, C481S
A01.01
YMANGSLLNY
175
13.24495
0.866167

BTK, C481S
A01.01
MANGSLLNY
171
439.029
0.216408

BTK, C481S
A03.01
MANGSLLNY
171
35.62463
0.237963

BTK, C481S
A03.01
YMANGSLLNY
175
95.93212
0.279088

BTK, C481S
A11.01
MANGSLLNY
171
535.6333
NB

BTK, C481S
A11.01
YMANGSLLNY
175
974.2881
NB

BTK, C481S
A24.02
EYMANGSLL
170
4.961145
5.716141

BTK_C481S
A02.01
SLLNYLREM
173
67.69132
3.043604

BTK_C481S
A02.01
MANGSLLNYL
172
1006.566
0

BTK_C481S
A02.01
YMANGSLLN
174
3999.442
0

BTK_C481S
B07.02
SLLNYLREM
173
865.8805
0

BTK_C481S
B07.02
MANGSLLNYL
172
16474.59
0

BTK_C481S
B08.01
SLLNYLREM
173
959.6542
0

BTK_C481S
B08.01
MANGSLLNYL
172
18463.09
0

Table 7 shows HLA affinity and stability of selected EGFR peptides:

TABLE 7

SEQ

Stability

HLA
Peptide
ID
Affinity
(half-life

Gene
Allele
Sequence
NO:
(nM)
(hr.))

EGFR, T790M
A01.01
LTSTVQLIM
182
2891.111
0.103721

EGFR_T790M
A01.01
CLTSTVQLIM
177
8276.876
0

EGFR_T790M
A02.01
MQLMPFGCLL
184
16.26147
0.381118

EGFR_T790M
A02.01
MQLMPFGCL
183
116.3352
0.368273

EGFR_T790M
A02.01
LIMQLMPFGC
181
132.4766
0.381284

EGFR_T790M
A02.01
QLIMQLMPF
185
192.8406
0.34067

EGFR_T790M
A02.01
CLTSTVQLIM
177
537.1391
0

EGFR_T790M
A02.01
IMQLMPFGCL
179
653.1065
0.515559

EGFR_T790M
A02.01
IMQLMPFGC
178
1205.368
0.370112

EGFR_T790M
A02.01
LIMQLMPFG
180
3337.708
0

EGFR_T790M
A02.01
VQLIMQLMPF
188
4942.892
0

EGFR_T790M
A02.01
QLIMQLMPFG
186
5214.668
0

EGFR_T790M
A02.01
STVQLIMQL
187
7256.773
0

EGFR_T790M
A24.02
QLIMQLMPF
185
2030.807
0.368673

EGFR_T790M
A24.02
VQLIMQLMPF
188
4103.131
0

EGFR_T790M
A24.02
IMQLMPFGCL
179
14119.38
0

EGFR_T790M
A24.02
MQLMPFGCLL
184
18857.47
0

EGFR_T790M
B07.02
MQLMPFGCL
183
1589.188
0

EGFR_T790M
B08.01
QLIMQLMPF
185
330.1933
0

EGFR_T790M
B08.01
IMQLMPFGCL
179
427.3913
0

EGFR_T790M
B08.01
MQLMPFGCL
183
4931.727
0

EGFR_T790M
B08.01
MQLMPFGCLL
184
11244.9
0

EGFR_T790M
B08.01
VQLIMQLMPF
188
16108.18
0

EGFR_T790M
B08.02
QLIMQLMPF
185
5590.3
ND

Tumor Antigens Associated with Tumor Microenvironment

In many cases, predominant antigens are expressed by cells in the tumor microenvironment that not only serve as excellent biomarkers for the disease, but also can be important vaccine candidates for immunotherapy. Such tumor associated antigens (TAAs) are not necessarily presented on the surface of tumor cells, but on cells that are juxtaposed to the tumor, which could be the stromal cells, connective tissue cells, fibroblasts etc. These are cells that often contribute to the structural integrity of the tumor, feed the tumor and support growth of the tumor. In most cases, TAAs are overexpressed antigens in the tumor microenvironment, however some antigens in the tumor microenvironment may also be unique in the tumor associated cells. As an example, telomerase reverse transcriptase (TERT) is a TAA that is not present in most normal tissues but is activated in most human tumors. Tissue kallikrein-related peptidases, or kallikreins (KLKs), on the other hand are overexpressed in various cancers and comprise a large family of secreted trypsin- or chymotrypsin-like serine proteases. Kallikreins are upregulated in prostrate ovarian and breast cancers. Some TAAs are specific to certain cancers, some are expressed in a large variety of cancers. Carcinoembryonic antigen (CEA) is overexpressed in breast, colon, lung and pancreatic carcinomas, whereas MUC-1 is breast, lung, prostate, colon cancers. Some TAAs are differentiation or tissue specific, for example, MART-1/melan-A and gp100 are expressed in normal melanocytes and melanoma, and prostate specific membrane antigen (PSMA) and prostate-specific antigen (PSA) are expressed by prostate epithelial cells as well as prostate carcinoma.

In some embodiments, T cells are developed for adoptive therapy that are directed to overexpressed tissue specific or tumor associated antigens, such as prostrate specific kallikrein proteins KLK2, KLK3, KLK4 in case of prostate cancer therapy, or transglutamase protein 4, TGM4 for adenocarcinoma.

In some embodiments, the antigenic peptides that are targeted for the adoptive therapy in the methods disclosed herein are effective in modulating the tumor microenvironment. T cells are primed with antigens expressed by cells in the TME, so that the therapy is directed towards weakening and/or breaking down the tumor facilitating TME, oftentimes, in addition to directly targeting the tumor cells for T cell mediated lysis.

Tumor microenvironment comprises fibroblasts, stromal cells, endothelial cells and connective tissue cells which make up a large proportion of cells that induce or influence tumor growth. Just as T cells can be stimulated and directed attack the tumor cells in a immunosuppressive tumor environment, certain peptides and antigens can be utilized to direct the T cells against cells in the tumor vicinity that help in tumor propagation CD8+ and CD4+ T cells can be generated ex vivo that are directed against antigens on the surface of non-tumor cells in the tumor microenvironment that promote tumor sustenance and propagation. Cancer/tumor associated fibroblasts (CAFs) are hallmark feature of pancreatic cancers, such as pancreatic adenocarcinoma (PDACs). CAFs express Col10a1 antigen. CAFs are cells that may help perpetuate a tumor. Col10A1 often confers negative prognosis for the tumor. In some embodiments Col10A1 may be considered as a biomarker for tumor sustenance and progression. It is a 680 amino acid long heterodimer protein associated with poor prognosis in breast cancer and colorectal cancers.

Activation of Col10a1 specific CD8+ T cells and CD4+ T cells may help attack and destruction of Col10A1 specific fibroblasts and help break down the tissue matrix of solid tumors.

T cells can be generated ex vivo using the method described herein, so that the T cells are activated against cancer-associated fibroblasts (CAFs). For this, Col10a1 peptides comprising epitopes that can specifically activate T cells were generated, and the HLA binding partner determined, using the highly reliable data generated from the in-house generated machine learning epitope presentation software described previously as described in Table 8.

TABLE 8

SEQ

Rank on

Peptide
ID NO:
HLA Allele
HLA allele

FTCQIPGIYY
1328
HLA-A01:01
1

GSDGKPGY
1329
HLA-A01:01
2

NAESNGLY
1330
HLA-A01:01
3

LTENDQVWL
1331
HLA-A01:01
4

GTHVWVGLY
1332
HLA-A01:01
5

TYDEYTKGY
1333
HLA-A01:01
6

YTYDEYTKGY
1334
HLA-A01:01
7

FTCQIPGIY
1335
HLA-A01:01
8

NAESNGLYSSEY
1336
HLA-A01:01
9

YLDQASGSA
1337
HLA-A01:01
10

FLLLVSLNL
1338
HLA-A02:01
1

FLLLVSLNLV
1339
HLA-A02:01
2

GLYKNGTPV
1340
HLA-A02:01
3

GLDGPKGNPGL
1341
HLA-A02:01
4

LLLVSLNLV
1342
HLA-A02:01
5

SLSGTPLVSA
1343
HLA-A02:01
6

GLYSSEYV
1344
HLA-A02:01
7

SLSGTPLV
1345
HLA-A02:01
8

MLPQIPFLL
1346
HLA-A02:01
9

GLPGPPGPSA
1347
HLA-A02:01
10

SAFTVILSK
1348
HLA-A03:01
1

AVMPEGFIK
1349
HLA-A03:01
2

GLYKNGTPVMY
1350
HLA-A03:01
3

AIGTPIPFDK
1351
HLA-A03:01
4

GLPGGPGAK
1352
HLA-A03:01
5

ILYNRQQHY
1353
HLA-A03:01
6

AGPPGPPGFGK
1354
HLA-A03:01
7

GIPGFPGSK
1355
HLA-A03:01
8

GTHVWVGLYK
1356
HLA-A03:01
9

GVPGQPGIK
1357
HLA-A03:01
10

AVMPEGFIK
1349
HLA-A11:01
1

SAFTVILSK
1348
HLA-A11:01
2

VSAFTVILSK
1358
HLA-A11:01
3

GTHVWVGLYK
1356
HLA-A11:01
4

AIGTPIPFDK
1351
HLA-A11:01
5

AVMPEGFIKA
1359
HLA-A11:01
6

SSFSGFLVA
1360
HLA-A11:01
7

PVSAFTVILSK
1361
HLA-A11:01
8

GIPGFPGSK
1355
HLA-A11:01
9

GVPGMNGQK
1362
HLA-A11:01
10

AYPAIGTPIPF
1363
HLA-A24:02
1

IGPPGIPGF
1364
HLA-A24:02
2

HYDPRTGIF
1365
HLA-A24:02
3

EYVHSSFSGF
1366
HLA-A24:02
4

AGPPGPPGF
1367
HLA-A24:02
5

YYFSYHVHV
1368
HLA-A24:02
6

AYPAIGTPI
1369
HLA-A24:02
7

PLPNTKTQF
1370
HLA-A24:02
8

MLPQIPFLL
1346
HLA-A24:02
9

CQIPGIYYF
1371
HLA-A24:02
10

RPSLSGTPL
1372
HLA-B07:02
1

LPQIPFLLL
1373
HLA-B07:02
2

IPFLLLVSL
1374
HLA-B07:02
3

LPGPPGPSAV
1375
HLA-B07:02
4

GPIGPPGIPGF
1376
HLA-B07:02
5

IPGPAGISV
1377
HLA-B07:02
6

YPAIGTPIPF
1378
HLA-B07:02
7

SPGPPGPAGI
1379
HLA-B07:02
8

LPGPPGPSA
1380
HLA-B07:02
9

SPGPPGPAG
1381
HLA-B07:02
10

TIKSKGIAV
1382
HLA-B08:01
1

IPFLLLVSL
1374
HLA-B08:01
2

HVHVKGTHV
1383
HLA-B08:01
3

LPNTKTQF
1384
HLA-B08:01
4

LPQIPFLL
1385
HLA-B08:01
5

PFLLLVSL
1386
HLA-B08:01
6

SLNLVHGV
1387
HLA-B08:01
7

LPQIPFLLL
1373
HLA-B08:01
8

TGMPVSAF
1388
HLA-B08:01
9

TPIPFDKIL
1389
HLA-B08:01
10

Neoantigenic peptides provided herein are prevalidated for HLA binding immunogenicity (Tables 1-8 and 11-14). In some embodiments the neoantigenic peptides, prepared and stored earlier, are used to contact an antigen presenting cell (APC) to then allow presentation to a T cell in vitro for preparation of neoantigen-specific activated T cell. In some embodiments, between 2-80 or more neoantigenic peptides are used to stimulate T cells from a patient at a time.

In some embodiments the APC is an autologous APC. In some embodiments the APC is a non-autologous APC. In some embodiments the APC is a synthetic cell designed to function as an APC. In some embodiments the T cell is an autologous cell. In some embodiments, an antigen presenting cell is a cell that expresses an antigen. For example, an antigen presenting cell may be a phagocytic cell such as a dendritic cell or myeloid cell, which process an antigen after cellular uptake and presents the antigen in association with an MEC for T cell activation. For certain purposes, an APC as used herein is a cell that normally presents an antigen on its surface. In a non-binding or non-limiting example, relevant to certain cytotoxicity assays as described herein, a tumor cell is an antigen presenting cell, that the T cell can recognize an antigen presenting cell (tumor cell). Similarly, a cell or cell line expressing an antigen can be, for certain purposes as used herein, an antigen presenting cell.

In some embodiments, one or more polynucleotides encoding one or more neoantigenic peptides may be used to express in a cell to present to a T cell for activation in vitro. The one or more polynucleotides encoding one or more of the neoantigenic peptides are encoded in a vector. In some embodiments, the composition comprises from about 2 to about 80 neoantigenic polynucleotides. In embodiments, at least one of the additional neoantigenic peptide is specific for an individual subject's tumor. In embodiments, the subject specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the subject's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample. In embodiments, the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells. In embodiments, the sequence differences are determined by Next Generation Sequencing.

In some embodiments the method and compositions provided herein can be used to identify or isolate a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein or an MEC-peptide complex comprising at least one neoantigenic peptide described herein. In embodiments, the MHC of the MHC-peptide is MHC class I or class II. In embodiments, TCR is a bispecific TCR further comprising a domain comprising an antibody or antibody fragment capable of binding an antigen. In embodiments, the antigen is a T cell-specific antigen. In embodiments, the antigen is CD3. In embodiments, the antibody or antibody fragment is an anti-CD3 scFv.

In some embodiments the method and compositions provided herein can be used to prepare a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding at least one neoantigenic peptide described herein or an MEC-peptide complex comprising at least one neoantigenic peptide described herein. In embodiments, CD3-zeta is the T cell activation molecule. In embodiments, the chimeric antigen receptor further comprises at least one costimulatory signaling domain. The In embodiments, the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI-gamma. In embodiments, the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MEW class I or class II. In embodiments, the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region. In embodiments, the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. In embodiments, the MHC of the MHC-peptide is MHC class I or class II.

Provided herein is a T cell comprising the T cell receptor or chimeric antigen receptor described herein, optionally wherein the T cell is a helper or cytotoxic T cell. In embodiments, the T cell is a T cell of a subject.

Provided herein is a T cell comprising a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein or an MHC-peptide complex comprising at least one neoantigenic peptide described herein, wherein the T cell is a T cell isolated from a population of T cells from a subject that has been incubated with antigen presenting cells and one or more of the at least one neoantigenic peptide described herein for a sufficient time to activate the T cells. In embodiments, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In embodiments, the population of T cells from a subject is a population of CD8+ T cells from the subject. In embodiments, the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide. In embodiments, the subject-specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject. In embodiments, the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not present in a non-tumor sample of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM. In embodiments, the activated CD8+ T cells are separated from the antigen presenting cells. In embodiments, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In embodiments, the antigen presenting cells are non-transformed cells. In embodiments, the antigen presenting cells are non-infected cells. In embodiments, the antigen presenting cells are autologous. In embodiments, the antigen presenting cells have been treated to strip endogenous MEC-associated peptides from their surface. In embodiments, the treatment to strip the endogenous MHC-associated peptides comprises culturing the cells at about 26° C. In embodiments, the treatment to strip the endogenous MEC-associated peptides comprises treating the cells with a mild acid solution. In embodiments, the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein. In embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 μg/mL of each of the at least one neoantigenic peptide described herein. In embodiments, ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. In embodiments, the incubating the isolated population of T cells is in the presence of IL-2 and IL-7. In embodiments, the MHC of the MHC-peptide is MHC class I or class II.

Provided herein is a method for activating tumor specific T cells comprising: isolating a population of T cells from a subject; and incubating the isolated population of T cells with antigen presenting cells and at least one neoantigenic peptide described herein for a sufficient time to activate the T cells. In embodiments, the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell. In embodiments, the population of T cells from a subject is a population of CD8+ T cells from the subject. In embodiments, the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide. In embodiments, the subject-specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject. In embodiments, the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not present in a non-tumor sample of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM. In embodiments, the method further comprises separating the activated T cells from the antigen presenting cells. In embodiments, the method further comprises testing the activated T cells for evidence of reactivity against at least one of neoantigenic peptide of described herein. In embodiments, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In embodiments, the antigen presenting cells are non-transformed cells. In embodiments, the antigen presenting cells are non-infected cells. In embodiments, the antigen presenting cells are autologous. In embodiments, the antigen presenting cells have been treated to strip endogenous MEC-associated peptides from their surface. In embodiments, the treatment to strip the endogenous MHC-associated peptides comprises culturing the cells at about 26° C. In embodiments, the treatment to strip the endogenous MEC-associated peptides comprises treating the cells with a mild acid solution. In embodiments, the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein. In embodiments, pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 μg/ml of each of at least one neoantigenic peptide described herein. In embodiments, ratio of isolated T cells to antigen presenting cells is between about 30:1 and 300:1. In embodiments, the incubating the isolated population of T cells is in the presence of IL-2 and IL-7. In embodiments, the MHC of the MHC-peptide is MHC class I or class II.

Provided herein is a composition comprising activated tumor specific T cells produced by a method described herein.

Provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of activated tumor specific T cell described herein, or produced by a method described herein. In embodiments, the administering comprises administering from about 10{circumflex over ( )}6 to 10{circumflex over ( )}12, from about 10{circumflex over ( )}8 to 10{circumflex over ( )}11, or from about 10{circumflex over ( )}9 to 10{circumflex over ( )}10 of the activated tumor specific T cells.

Provided herein is a nucleic acid comprising a promoter operably linked to a polynucleotide encoding the T cell receptor described herein. In embodiments, the TCR is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II.

Provided herein is a nucleic acid comprising a promoter operably linked to a polynucleotide encoding the chimeric antigen receptor described herein. In embodiments, the antigen recognition moiety is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In embodiments, the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide. In embodiments, the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.

In some embodiments the autologous immune cells from the peripheral blood of the patient constitute peripheral blood mononuclear cells (PBMC). In some embodiments the autologous immune cells from the peripheral blood of the patient are collected via an apheresis procedure. In some embodiments, the PBMCs are collected from more than one apheresis procedures, or more than one draw of peripheral blood.

In some embodiments, both CD25+ cells and the CD14+ cells are depleted prior to addition of peptides. In some embodiments, either of CD25+ cells or the CD14+ cells are depleted prior to addition of peptides. In some embodiments, CD25+ cells and not the CD14+ cells are depleted prior to addition of peptides.

In some embodiments, the depletion procedure is followed by the addition of FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to stimulate the antigen presenting cells (APCs), constituted by the monocytes, macrophages or dendritic cells (DCs) prior to addition of the peptides. In some embodiments, the depletion procedure is followed by selection of DC as suitable PACs for peptide presentation to the T cells, and mature macrophages and other antigen presenting cells are removed from the autologous immune cells from the patient. In some embodiments, the depletion procedure is followed by selection of immature DC as suitable PACs for peptide presentation to the T cells.

In some embodiments, a selection of ‘n’ number of neoantigenic peptides is contacted with the APCs for stimulation of the APCs for antigen presentation to the T cells.

In some embodiments, a first level selection of ‘n’ number of neoantigenic peptides is based on the binding ability of each of the peptides to at least on HLA haplotype that is predetermined to be present in the recipient patient. In order to determine HLA haplotype that is predetermined to be present in the recipient patient, as is known to one of skill in the art, a patient is subjected to HLA haplotyping assay form a blood sample prior to the commencement of the treatment procedure. In some embodiments, a first level selection of ‘n’ number of neoantigenic peptides is followed by a second level selection based on the determination of whether the mutation present in the neoantigenic peptide(s) match the neoantigens (or mutations leading to) known to be found in at least 5% of patients known to have the cancer. In some embodiments, the second level of the selection involves further determination of whether the mutation is evident in the patient.

In some embodiments, a first and the second level selection of ‘n’ number of neoantigenic peptides for contacting the APCs is followed by a third level of selection, based on the binding affinity of the peptide with the HLA that the peptide is capable of binding to and is at least less than 500 nM, with the determination that higher the binding affinity, the better the choice of the peptide to be selected. In some embodiments, the finally selected ‘n’ number of peptides can range from 1-200 peptides which are in a mix, for exposing APCs to the peptides in the culture media, and contacting with APCs.

In some embodiments the ‘n’ number of peptides can range from 10-190 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 20-180 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 30-170 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 40-160 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 50-150 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 60-140 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 70-130 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 80-120 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 50-100 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 50-90 neoantigenic peptides. In some embodiments the ‘n’ number of peptides can range from 50-80 neoantigenic peptides. In some embodiments the ‘n’ number of peptides comprise at least 60 neoantigenic peptides. In some embodiments the ‘n’ number of peptides comprise a mixture of (a) neoantigenic peptides that are short, 8-15 amino acids long, comprising the mutated amino acid as described previously, following the formula AxByCz; these peptides are interchangeably called shortmers or short peptides for the purpose of this application; and (b) long peptides that are 15, 30, 50, 60, 80, 100-300 amino acids long and any length in between, which are subject to endogenous processing by dendritic cells for better antigen presentation; these peptides are interchangeably called longmers or long peptides for the purpose of this application. In some embodiments the at least 60 neoantigenic peptides comprise at least 30 shortmers and at least 30 longmers or variations of the same. Exemplary variations of the same include, but are not limited to the following: in some embodiments the at least 60 neoantigenic peptides comprise at least 32 shortmers and at least 32 longmers or variations of the same. In some embodiments the at least 60 neoantigenic peptides comprise at least 34 shortmers and at least 30 longmers or variations of the same. In some embodiments the at least 60 neoantigenic peptides comprise at least 28 shortmers and at least 34 longmers or variations of the same.

In some embodiments, the ‘n’ number of peptides are incubated in the medium comprising APCs in culture, where the APCs (DCs) have been isolated from the PBMCs, and previously stimulated with FLT3L. In some embodiments, the ‘n’ number of peptides are incubated with APCs in presence of FLT3L. In some embodiments, following the step of incubation of the APCs with FLT3L, the cells are added with fresh media containing FL3TL for incubation with peptides. In some embodiments, the maturation of APCs to mature peptide loaded DCs may comprise several steps of culturing the DCs towards maturation, examining the state of maturation by analysis of one or more released substances, (e.g. cytokines, chemokines) in the culture media or obtaining an aliquot of the DCs in culture form time to time. In some embodiments, the maturation of DCs take at least 5 days in culture from onset of the culture. In some embodiments, the maturation of DCs take at least 7 days in culture from onset of the culture. In some embodiments, the maturation of DCs take at least 11 days in culture from onset of the culture, or any number of days in between.

In some embodiments, the DCs are contacted with T cells after being verified for presence of or absence of maturation factors and peptide tetramer assay for verifying the repertoire of antigens presented.

In some embodiments, the DCs are contacted with T cells in a T cell media for about 2 days for the first induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 3 days for the first induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 4 days for the first induction. In some embodiments, the DCs are contacted with T cells in a T cell media for at least about 2 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for at least about 3 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for at least about 4 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for 5 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 6 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 7, 8, 9 or 10 days for the second induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about less than 1 days for the third induction. In some embodiments, the DCs are contacted with T cells in a T cell media for at least about 2 or 3 days for the third induction. In some embodiments, the DCs are contacted with T cells in a T cell media for at least about 4 days for the third induction. In some embodiments, the DCs are contacted with T cells in a T cell media for 5 days for the third induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 6 days for the third induction. In some embodiments, the DCs are contacted with T cells in a T cell media for about 7, 8, 9 or 10 days for the second induction.

In some embodiments, the T cells are further contacted with one or more shortmer peptides during incubation with DCs (and in addition to the DCs) at either the first induction phase, the second induction phase or the third induction phase. In some embodiments, the T cells are further contacted with one or more shortmer peptides during incubation with DCs at the first induction phase and the second induction phase. In some embodiments, the T cells are further contacted with one or more shortmer peptides during incubation with DCs at the second induction phase and the third induction phase. In some embodiments, the T cells are further contacted with one or more shortmer peptides in all the three induction phases.

In some embodiments, the APCs and the T cells are comprised in the same autologous immune cells from the peripheral blood of the patient drawn at the first step from the patient. The T cells are isolated and preserved for the time of activation with the DCs at the end of the DC maturation phase. In some embodiments the T cells are cocultured in the presence of a suitable media for activation for the time of activation with the DCs at the end of the DC maturation phase. In some embodiments the T cells are prior cyropreserved cells from the patient, which are thawed and cultured for at least 4 hours to up to about 48 hours for induction at the time of activation with the DCs at the end of the DC maturation phase.

In some embodiments, the APCs and the T cells are comprised in the same autologous immune cells from the peripheral blood of the patient drawn at the different time periods from the patient, e.g. at different apheresis procedures. In some embodiments the time from apheresis of the patient to the time of harvest, takes between about 20 days to about less than 26 days. In some embodiments the time from apheresis of the patient to the time of harvest, takes between about 21 days to about less than 25 days. In some embodiments the time from apheresis of the patient to the time of harvest, takes between about 21 days to about less than 24 days. In some embodiments the time from apheresis of the patient to the time of harvest, takes between about 21 days to about less than 23 days. In some embodiments the time from apheresis of the patient to the time of harvest, takes about 21 days. In some embodiments the time from apheresis of the patient to the time of harvest, takes about less than 21 days.

In some embodiments the release criteria for the activated T cells (the drug substance) comprises any one or more of sterility, endotoxin, cell phenotype, TNC Count, viability, cell concentration, potency. In some embodiments the release criteria for the activated T cells (the drug substance) comprises each one of sterility, endotoxin, cell phenotype, TNC Count, viability, cell concentration, potency.

In some embodiments the total number of cells is 2×10{circumflex over ( )}10. In some embodiments the total number of cells is 2×10{circumflex over ( )}9. In some embodiments the total number of cells is 5×10{circumflex over ( )}8. In some embodiments the total number of cells is 2×10{circumflex over ( )}8. In some embodiments the final concentration of the resuspended T cells is 2×10{circumflex over ( )}5 cells/ml or more. In some embodiments the final concentration of the resuspended T cells is 1×10{circumflex over ( )}6 cells/ml or more. In some embodiments the final concentration of the resuspended T cells is 2×10{circumflex over ( )}6 cells/ml or more.

The following criteria of released cells are described as exemplary non-limiting conditions, particularly because of the reason that the criteria for the cell population and subpopulations in Drug substance (DS) can vary based on the cancer, the state of the cancer, the state of the patient, the availability of the matched HLA haplotype and the growth potential of the APCs and T cells in the presence of the peptide. In some embodiments the activated T cells (the drug substance) comprises at least 2% or at least 3% or at least 4% or at least 5% of CD8+ T cells reactive to a particular neoantigen by tetramer assay. In some embodiments, the activated T cells (the drug substance) comprises at least 2% or at least 3% or at least 4% or at least 5% of CD4+ T cells reactive to a particular neoantigen by tetramer assay. In some embodiments, the activated T cells (the drug substance) comprise at least 5% or at least 6% or at least 7% or at least 8% or at least 9% or at least 10% of cells that are positive for memory T cell phenotype.

In some embodiments, the activated T cells (the drug substance) are selected based on one or more markers. In some embodiments, the activated T cells (the drug substance) are not selected based on one or more markers. In some embodiments, an aliquot of the activated T cells (the drug substance) are tested for the presence or absence of one or more of the following markers, and the proportions of cells thereof exhibiting each of the tested markers, the one or more markers are selected from a group consisting of: CD19, CD20, CD21, CD22, CD24, CD27, CD38, CD40, CD72, CD3, CD79a, CD79b, IGKC, IGHD, MZB1, TNFRSF17, MS4A1, CD138, TNFRSR13B, GUSPB11, BAFFR, AID, IGHM, IGHE, IGHA1, IGHA2, IGHA3, IGHA4, BCL6, FCRLA CCR7, CD27, CD45RO, FLT3LG, GRAP2, IL16, IL7R, LTB, S1PR1, SELL, TCF7, CD62L, PLACE, SORL1, MGAT4A, FAM65B, PXN, A2M, ATM, C20orf112, GPR183, EPB41, ADD3, GRAP2, KLRG1, GIMAP5, TC2N, TXNIP, GIMAP2, TNFAIP8, LMNA, NR4A3, CDKN1A, KDM6B, ELL2, TIPARP, SC5D, PLK3, CD55, NR4A1, REL, PBX4, RGCC, FOSL2, SIK1, CSRNP1, GPR132, GLUL, KIAA1683, RALGAPA1, PRNP, PRMT10, FAM177A1, CHMP1B, ZC3H12A, TSC22D2, P2RY8, NEU1, ZNF683, MYADM, ATP2B1, CREM, OAT, NFE2L2, DNAJB9, SKIL, DENND4A, SERTAD1, YPEL5, BCL6, EGR1, PDE4B, ANXA1, SOD2, RNF125, GADD45B, SELK, RORA, MXD1, IFRD1, PIK3R1, TUBB4B, HECA, MPZL3, USP36, INSIG1, NR4A2, SLC2A3, PER1, S100A10, AIM1, CDC42EP3, NDEL1, IDI1, EIF4A3, BIRC3, TSPYL2, DCTN6, HSPH1, CDK17, DDX21, PPP1R15B, ZNF331, BTG2, AMD1, SLC7A5 POLR3E, JMJD6, CHD1, TAF13, VPS37B, GTF2B, PAF1, BCAS2, RGPD6, TUBA4A, TUBA1A, RASA3, GPCPD1, RASGEF1B, DNAJA1, FAM46C, PTP4A1, KPNA2, ZFAND5, SLC38A2, PLIN2, HEXIM1, TMEM123, JUND, MTRNR2L1, GABARAPL1, STAT4, ALG13, FOSB, GPR65, SDCBP, HBP1, MAP3K8, RANBP2, FAM129A, FOS, DDIT3, CCNH, RGPD5, TUBA1C, ATP1B3, GLIPR1, PRDM2, EMD, HSPD1, MORF4L2, IL21R, NFKBIA, LYAR, DNAJB6, TMBIM1, PFKFB3, MED29, B4GALT1, NXF1, BIRC2, ARHGAP26, SYAP1, DNTTIP2, ETF1, BTG1, PBXIP1, MKNK2, DEDD2, AKIRIN1, HLA-DMA, HLA-DNB, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, CCL18, CCL19, CCL21, CXCL13, LAMP3, LTB, IL7R, MS4A1, CCL2, CCL3, CCL4, CCL5, CCL8, CXCL10, CXCL11, CXCL9, CD3, LTA, IL17, IL23, IL21, IL7, CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2, PSMB10, STAT1, TIGIT, CD56, CCL2, CCL3, CCL4, CCL5, CXCL8, IFN, IL-2, IL-12, IL-15, IL-18, NCR1, XCL1, XCL2, IL21R, KIR2DL3, KIR3DL1, KIR3DL2, NCAM1, HLA-DMA, HLA-DNB, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5.

In some embodiments, at least 0.01% of naive T cells which were obtained from the obtaining of autologous immune cells from the peripheral blood of the patient were stimulated in response to a neoantigen, and was amplified at the end of the procedure and was harvested. In some embodiments, greater than 0.01% of naive T cells which were obtained from the obtaining of autologous immune cells from the peripheral blood of the patient were stimulated in response to a neoantigen, and was amplified at the end of the procedure and was harvested. In some embodiments, greater than 0.1% of naive T cells which were obtained from the obtaining of autologous immune cells from the peripheral blood of the patient were stimulated in response to a neoantigen, and was amplified at the end of the procedure and was harvested. In some embodiments, greater than 1% of naive T cells which were obtained from the obtaining of autologous immune cells from the peripheral blood of the patient were stimulated in response to a neoantigen, and was amplified at the end of the procedure and was harvested.

In some embodiments the total number of cells is harvested from 1, 2, or 3 cycles of the process of DC maturation and T cell activation.

In some embodiments the harvested cells are cryopreserved in vapor phase of liquid nitrogen in bags.

As is known to one of skill in the art, all applications described in the preceding paragraphs of this section from obtaining of autologous immune cells from the peripheral blood of the patient to the harvesting of cells is performed in an aseptic closed system, except the steps where aliquots of media or cells are taken out for examination by flow cytometry, mass spectroscopy, cell count, cell sorting or any functional assays, that are terminal to the cells or materials taken out as aliquots. In some embodiments the closed system for aseptic culture of up to the harvesting is proprietary to the applicant's process.

In some embodiments the T cells are method for culturing and expansion of activated T cells including the steps delineated above, starting from obtaining of autologous immune cells from the peripheral blood of the patient to harvesting, is scalable in an aseptic procedure. In some embodiments, at least 1 Liter of DC cell culture is performed at a time. In some embodiments, at least 1-2 Liters of T cell culture is performed at a time. In some embodiments, at least 5 Liters of DC cell culture is performed at a time. In some embodiments, at least 5-10 Liters of T cell culture is performed at a time. In some embodiments, at least 10 Liter of DC cell culture is performed at a time. In some embodiments, at least 10-40 Liters of T cell culture is performed at a time. In some embodiments, at least 10 Liter of DC cell culture is performed at a time. In some embodiments, at least 10-50 Liters of T cell culture is performed at a time. In some embodiments, simultaneous batch cultures are performed and tested in a system that is a closed system, and that can be manipulated and intervened from outside without introducing non-aseptic means. In some embodiments, a closed system described herein is fully automated.

When administration is by injection, the active agent can be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. The solution can contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active compound can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In another embodiment, the drug product comprises a substance that further activates or inhibits a component of the host's immune response, for example, a substance to reduce or eliminate the host's immune response to the peptide.

The disclosure provided herein demonstrates that shared neoantigens can be used for ready therapeutic administration of a patient, thereby reducing the bench-to-bedside time lag considerably. The composition and methods described herein provide innovative advancements in the field of cancer therapeutics.

EXAMPLES
Example 1. Precision NEOSTIM Clinical Process

Provided herein is an adoptive T cell therapy where T cells primed and responsive against curated pre-validated, shelved, antigenic peptides specific for a subject's cancer is administered to the subject. Provided in this example is a method of bypassing lengthy sequencing, identification and manufacture of subject specific neoantigen peptides and thereafter generating T cells having the subject specific TCRs for cancer immunotherapy, at least for the time when a subject undergoes a process of such evaluation and preparations for the personalized therapy. Advantage of this process is that it is fast, targeted and robust. As shown in FIG. 1A, patient identified with a cancer or tumor can be administered T cells that are activated ex vivo with warehouse curated peptides having selected, prevalidated collection of epitopes generated from a library of shared antigens known for the identified cancer. The process from patient selection to the T cell therapy may require less than 6 weeks. FIG. 1B illustrates the method of generating cancer target specific T cells ex vivo by priming T cells with antigen presenting cells (APCs) expressing putative T cell epitopes and expanding the activated T cells to obtain epitope-specific CD8+ and CD4+ including a population of these cells exhibiting memory phenotype (see, e.g., WO2019094642, incorporated by reference in its entirety). A library of prevalidated epitopes is generated in advance. Such epitopes are collected from prior knowledge in the field, common driver mutations, common drug resistant mutations, tissue specific antigens, and tumor associated antigens. With the help of an efficient computer-based program for epitope prediction, HLA binding and presentation characteristics, pre-validated peptides are generated for storage and stocking as shown in a diagram in FIG. 2. Exemplary predictions for common RAS G12 mutations are shown in FIG. 3A-3D. Validations are performed using a systematic process as outlined in Examples 2-5. Target tumor cell antigen responsive T cells are generated ex vivo and immunogenicity is validated using an in vitro antigen-specific T cell assay (Example 2). Mass spectrometry is used to validate that cells that express the antigen of interest can process and present the peptides on the relevant HLA molecules (Example 3). Additionally, the ability of these T cells to kill cells presenting the peptide is confirmed using a cytotoxicity assay (Example 4). Exemplary data provided herein demonstrate this validation process for RAS and GATA3 neoantigens, and can be readily applied to other antigens.

Example 2. Generation of Target Tumor Cell Antigen Responsive T Cells Ex Vivo

Materials:

AIM V media (Invitrogen)

Human FLT3L, preclinical CellGenix #1415-050 Stock 50 ng/μL

TNF-α, preclinical CellGenix #1406-050 Stock 10 ng/μL

IL-1β, preclinical CellGenix #1411-050 Stock 10 ng/μL

PGE1 or Alprostadil—Cayman from Czech republic Stock 0.5 μg/μL

R10 media—RPMI 1640 glutamax+10% Human serum+1% PenStrep

20/80 Media—18% AIM V+72% RPMI 1640 glutamax+10% Human Serum+1% PenStrep

IL7 Stock 5 ng/μL

IL15 Stock 5 ng/μL

Procedure:

Step 1: Plate 5 million PBMCs (or cells of interest) in each well of 24 well plate with FLT3L in 2 mL AIM V media

Step 2: Peptide loading and maturation—in AIMV

1. Mix peptide pool of interest (except for no peptide condition) with PBMCs (or cells of interest) in respective wells.

2. Incubate for 1 hr.

3. Mix Maturation cocktail (including TNF-α, IL-1β, PGE1, and IL-7) to each well after incubation.

Step 3: Add human serum to each well at a final concentration of 10% by volume and mix.

Step 4: Replace the media with fresh RPMI+10% HS media supplemented with IL7+IL15.

Step 5: Replace the media with fresh 20/80 media supplemented with IL7+IL15 during the period of incubation every 1-6 days.

Step 6: Plate 5 million PBMCs (or cells of interest) in each well of new 6-well plate with FLT3L in 2 ml AIM V media

Step 7: Peptide loading and maturation for re-stimulation—(new plates)

1. Mix peptide pool of interest (except for no peptide condition) with PBMCs (or cells of interest) in respective wells

2. Incubate for 1 hr.

3. Mix Maturation cocktail to each well after incubation

Step 8: Re-stimulation:

1. Count first stimulation FLT3L cultures and add 5 million cultured cells to the new Re-stimulation plates.

2. Bring the culture volume to 5 mL (AIM V) and add 500 μL of Human serum (10% by volume)

Step 9: Remove 3 ml of the media and add 6 ml of RPMI+10% HS media supplemented with IL7+IL15.

Step 10: Replace 75% of the media with fresh 20/80 media supplemented with IL7+IL15.

Step 11: Repeat re-stimulation if needed.

Analysis of Antigen-Specific Induction

MHC tetramers are purchased or manufactured on-site according to methods known by one of ordinary skill, and are used to measure peptide-specific T cell expansion in the immunogenicity assays. For the assessment, tetramer is added to 1×10⁵cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) according to manufacturer's instructions. Cells are incubated in the dark for 20 minutes at room temperature. Antibodies specific for T cell markers, such as CD8, are then added to a final concentration suggested by the manufacturer, and the cells are incubated in the dark at 4° C. for 20 minutes. Cells are washed with cold FACS buffer and resuspended in buffer containing 1% formaldehyde. Cells are acquired on a LSR Fortessa (Becton Dickinson) instrument, and are analyzed by use of FlowJo software (Becton Dickinson). For analysis of tetramer positive cells, the lymphocyte gate is taken from the forward and side-scatter plots. Data are reported as the percentage of cells that were CD8⁺/tetramer⁺.

Exemplary data for RAS neoantigens on HLA-A03:01 and HLA-A11:01 are shown in FIG. 5. Exemplary data across multiple healthy donors for RAS G12V neoantigens on HLA-A11:01 are shown in FIG. 6. Exemplary data for RAS G12V neoantigens on HLA-A02:01 are shown in FIG. 13. Exemplary data for RAS neoantigens on HLA-A68:01 are shown in FIG. 14. Exemplary data for RAS neoantigens on HLA-B07:02 are shown in FIG. 15. Exemplary data for RAS neoantigens on HLA-B08:01 are shown in FIG. 16. Exemplary data for a RAS G12D neoantigens on HLA-008:02 are shown in FIG. 17. Exemplary data for GATA3 neoantigens on HLA-A02:01, HLA-A03:01, HLA-A11:01, HLA-B07:02, and HLA-B08:01 are shown in FIG. 21. Exemplary data for a BTK C481S neoantigen on HLA-A02:01 are shown in FIG. 26. Exemplary data for EGFR T790M neoantigens on HLA-A02:01 are shown in FIG. 27.

CD4⁺ T cell responses towards neoantigens can be induced using the ex vivo induction protocol. In this example, CD4⁺ T cell responses were identified by monitoring IFNγ and/or TNFα production in an antigen specific manner. FIG. 18 shows representative examples of such flow cytometric analysis for CD4+ T cells reactive to a RAS G12D neoantigen. FIG. 24 shows representative examples of such flow cytometric analysis for CD4+ T cells reactive to a GATA3 neoantigen.

Example 3. Evaluation of Presentation of Antigens

For a subset of predicted antigens, the affinity of the neoepitopes for the indicated HLA alleles and stability of the neoepitopes with the HLA alleles was determined. Exemplary data for a subset of RAS neoantigens and GATA3 neoantigens are shown in FIGS. 4A and 20, respectively.

An exemplary detailed description of the protocol utilized to measure the binding affinity of peptides to Class I MHC has been published (Sette et al, Mol. Immunol. 31(11):813-22, 1994). In brief, MHCI complexes were prepared and bound to radiolabeled reference peptides. Peptides were incubated at varying concentrations with these complexes for 2 days, and the amount of remaining radiolabeled peptide bound to WWI was measured using size exclusion gel-filtration. The lower the concentration of test peptide needed to displace the reference radiolabeled peptide demonstrates a stronger affinity of the test peptide for MHCI. Peptides with affinities to MHCI<50 nM are generally considered strong binders while those with affinities <150 nM are considered intermediate binders and those <500 nM are considered weak binders (Fritsch et al, 2014).

An exemplary detailed description of the protocol utilized to measure the binding stability of peptides to Class I MHC has been published (Harndahl et al. J Immunol Methods. 374:5-12, 2011). Briefly, synthetic genes encoding biotinylated MHC-I heavy and light chains are expressed in E. coli and purified from inclusion bodies using standard methods. The light chain (β2m) is radio-labeled with iodine (125I), and combined with the purified MHC-I heavy chain and peptide of interest at 18° C. to initiate pMHC-I complex formation. These reactions are carried out in streptavidin coated microplates to bind the biotinylated MHC-I heavy chains to the surface and allow measurement of radiolabeled light chain to monitor complex formation. Dissociation is initiated by addition of higher concentrations of unlabeled light-chain and incubation at 37° C. Stability is defined as the length of time in hours it takes for half of the complexes to dissociate, as measured by scintillation counts.

To assess whether antigens could be processed and presented from the larger polypeptide context, peptides eluted from HLA molecules isolated from cells expressing the genes of interest were analyzed by tandem mass spectrometry (MS/MS).

For analysis of presentation of RAS neoantigens, cell lines were utilized that have RAS mutations naturally or were lentivirally transduced to express the mutated RAS gene. HLA molecules were either isolated based on the natural expression of the cell lines or the cell lines were lentivirally transduced or transiently transfected to express the HLA of interest. 293T cells were transduced with a lentiviral vector encoding various regions of a mutant RAS peptide. Greater than 50 million cells expressing peptides encoded by a mutant RAS peptide were cultured and peptides were eluted from HLA-peptide complexes using an acid wash. Eluted peptides were then analyzed by targeted MS/MS with parallel reaction monitoring (PRM). For 293T cells lentivirally transduced with both a RAS^G12Vmutation and an HLA-A*03:01 gene, the peptide with amino acid sequence vvvgaVgvgk (SEQ ID NO: 5) was detected by mass spectrometry. Spectral comparison to its corresponding stable heavy-isotope labeled synthetic peptide (FIG. 4B) showed mass accuracy of the detected peptide to be less than 5 parts per million (ppm). Endogenous peptide spectra are shown in the top panels and corresponding stable heavy-isotope labeled spectra are shown in the bottom panels. For SW620 cells naturally expressing a RAS^G12Vmutation and lentivirally transduced with the HLA-A*03:01 gene, the peptide with amino sequence vvvgaVgvgk (SEQ ID NO: 5) was detected by mass spectrometry. Spectral comparison to its corresponding stable heavy-isotope labeled synthetic peptide showed mass accuracy of the detected peptide to be less than 5 ppm (FIG. 4C). Endogenous peptide spectra are shown in the top panels and corresponding stable heavy-isotope labeled spectra are shown in the bottom panels. For NCI-H441 cells naturally expressing both the RAS^G12Vmutation and the HLA-A*03:01 gene, the peptide with amino acid sequence vvvgaVgvgk (SEQ ID NO: 5) was detected by mass spectrometry. Spectral comparison to its corresponding stable heavy-isotope labeled synthetic peptide showed mass accuracy of the detected peptide to be less than 5 ppm (FIG. 4D). Endogenous peptide spectra are shown in the top panels and corresponding stable heavy-isotope labeled spectra are shown in the bottom panels. A similar procedure was performed to analyze peptides derived from multiple RAS^G12mutations on HLA-A*03:01, HLA-A*11:01, HLA-A*30:01, HLA-A*68:01 and HLA-B*07:02 and Table 13 lists those peptides that were detected by mass spectrometry.

TABLE 13

SEQ

Allele
Mutation
Neoantigen
ID NO:
Length

A*03:01
G12C
vvvgaCgvgk
157
10

G12D
vvvgaDgvgk
159
10

G12D
klvvvgaDgvgk
1252
12

G12R
vvvgaRgvgk
3
10

G12R
klvvvgaRgvgk
1262
12

G12V
vvvgaVgvgk
5
10

G12V
vvgaVgvgk
164
9

G12V
klvvvgaVgvgk
1283
12

A*11:01
G12C
vvvgaCgvgk
157
10

G12D
vvvgaDgvgk
159
10

G12R
vvvgaRgvgk
3
10

G12V
vvvgaVgvgk
5
10

G12V
vvgaVgvgk
164
9

A*30:01
G12R
vvvgaRgvgk
3
10

A*68:01
G12C
vvvgaCgvgk
157
10

G12D
vvvgaDgvgk
159
10

G12R
vvvgaRgvgk
3
10

G12V
vvvgaVgvgk
5
10

G12V
vvgaVgvgk
164
9

G12V
lvvvgaVgvgk
1282
11

B*07:02
G12D
gaDgvgksal
1244
10

G12R
gaRgvgksal
1255
10

For analysis of presentation of GATA3 neoantigens, 293T cells were transduced with a lentiviral vector encoding various regions of peptides encoded by the GATA3 neoORF. Between 50 and 700 million of the transduced cells expressing peptides encoded by the GATA3 neoORF sequence were cultured and peptides were eluted from HLA-peptide complexes using an acid wash. Eluted peptides were then analyzed by targeted MS/MS using PRM. Spectral comparison between peptides derived from GATA3 neoORF and corresponding synthetic peptides were performed to confirm each detection. For 293T cells expressing an HLA-A*02:01 protein, the peptides VLPEPHLAL (SEQ ID NO: 1084), SMLTGPPARV (SEQ ID NO: 6) and MLTGPPARV (SEQ ID NO: 1081) were detected by mass spectrometry (Table 14 and FIG. 20). Spectral comparison to corresponding synthetic peptides showed mass accuracy of the detected peptide (SMLTGPPARV (SEQ ID NO: 6)) to be less than 5 ppm (FIG. 4E). For 293T cells expressing an HLA-A*03:01 or HLA-A*11:01 protein, the peptide KIMFATLQR (SEQ ID NO: 1089) was detected by mass spectrometry (FIG. 20). For 293T cells expressing an HLA-A*30:02 protein, the peptides IMKPKRDGY (SEQ ID NO: 1390) and SIMKPKRDGY (SEQ ID NO: 1391) were detected by mass spectrometry (Table 14). For 293T cells expressing an HLA-B*07:02 protein, the peptides KPKRDGYMF (SEQ ID NO: 1093) and KPKRDGYMFL (SEQ ID NO: 1095) were detected by mass spectrometry (Table 14 and FIG. 20). For 293T cells expressing an HLA-B*08:01 protein, the peptide ESKIMFATL (SEQ ID NO: 1091) was detected by mass spectrometry (Table 14 and FIG. 20). For 293T cells expressing an HLA-B*40:02 protein, the peptides AESKIMFATL (SEQ ID NO: 1392) and AESKIMFAT (SEQ ID NO: 1393) were detected by mass spectrometry (Table 14). For 293T cells expressing an HLA-C*03:03 protein, the peptide FATLQRSSL (SEQ ID NO: 1078) was detected by mass spectrometry (Table 14).

TABLE 14

SEQ ID

Allele
Mutation
Neoantigen
NO:
Length

A*02:01
GATA3 neoORF
VLPEPHLAL
1084
9

GATA3 neoORF
SMLTGPPARV
6
10

GATA3 neoORF
MLTGPPARV
1081
9

A*03:01
GATA3 neoORF
KIMFATLQR
1089
9

A*11:01
GATA3 neoORF
KIMFATLQR
1089
9

A*30:02
GATA3 neoORF
EVIKPKRDGY
1390
9

GATA3 neoORF
SIMKPKRDGY
1391
10

B*07:02
GATA3 neoORF
KPKRDGYMF
1093
9

GATA3 neoORF
KPKRDGYMFL
1095
10

B*08:01
GATA3 neoORF
ESKIMFATL
1091
9

B*40:02
GATA3 neoORF
AESKIMFATL
1392
10

GATA3 neoORF
AESKIMFAT
1393
9

C*03:03
GATA3 neoORF
FATLQRSSL
1078
9

HLA Class I Binding and Stability

A subset of the peptides used for affinity measurements were also used for stability measurements using the assay described (n=275). These data are shown in Table 3. Less than 50 nM was considered by the field as a strong binder, 50-150 nM was considered an intermediate binder, 150-500 nM was considered a weak binder, and greater than 500 nM was considered a very weak binder. The connection between the observed stability and observed affinity was evident by the decreasing median stability across these binned stability intervals. However, there is considerable overlap between the bins, and importantly there are epitopes in all bins with observed stability in the multiple hour range, including the very weak binders.

Immunogenicity assays are used to test the ability of each test peptide to expand T cells. Mature professional APCs are prepared for these assays in the following way. Monocytes are enriched from healthy human donor PBMCs using a bead-based kit (Miltenyi). Enriched cells are plated in GM-CSF and IL-4 to induce immature DCs. After 5 days, immature DCs are incubated at 37° C. with each peptide for 1 hour before addition of a cytokine maturation cocktail (GM-CSF, IL-1β, IL-4, IL-6, TNFα, PGE1β). Cells are incubated at 37° C. to mature DCs. In some embodiments the peptides, when administered into a patient is required to elicit an immune response.

Table 4A shows peptide sequences comprising RAS mutations, corresponding HLA allele to which it binds, and measured stability and affinity.

Example 4. Assessment of Cytotoxic Capacity of Antigen-Specific T Cells In Vitro

Cytotoxicity activity can be measured with the detection of cleaved Caspase 3 in target cells by Flow cytometry. Target cancer cells are engineered to express the mutant peptide along and the proper MHC-I allele. Mock-transduced target cells (i.e. not expressing the mutant peptide) are used as a negative control. The cells are labeled with CFSE to distinguish them from the stimulated PBMCs used as effector cells. The target and effector cells are co-cultured for 6 hours before being harvested. Intracellular staining is performed to detect the cleaved form of Caspase 3 in the CFSE-positive target cancer cells. The percentage of specific lysis is calculated as: Experimental cleavage of Caspase 3/spontaneous cleavage of Caspase 3 (measured in the absence of mutant peptide expression)×100. Exemplary data showing that T cells induced against GATA3 neoantigens can kill target cells expressing the GATA3 neoORF is shown in FIG. 23.

In some examples, cytotoxicity activity is assessed by co-culturing induced T cells with a population of antigen-specific T cells with target cells expressing the corresponding HLA, and by determining the relative growth of the target cells, along with measuring the apoptotic marker Annexin V in the target cancer cells specifically. Target cancer cells are engineered to express the mutant peptide or the peptide is exogenously loaded. Mock-transduced target cells (i.e. not expressing the mutant peptide), target cells loaded with wild-type peptides, or target cells with no peptide loaded are used as a negative control. The cells are also transduced to stably express GFP allowing the tracking of target cell growth. The GFP signal or Annexin-V signal are measured over time with an IncuCyte S3 apparatus. Annexin V signal originating from effector cells is filtered out by size exclusion. Target cell growth and death is expressed as GFP and Annexin-V area (mm²) over time, respectively.

Exemplary data demonstrating that T cells stimulated to recognize a RAS^G12Vneoantigen on HLA-A11:01 specifically recognize and kill target cells loaded with the mutant peptide but not the wild-type peptide is shown in FIG. 7. Exemplary data demonstrating that T cells stimulated to recognize a RAS^G12Vneoantigen on HLA-A11:01 kill target cells loaded with nanomolar amounts of peptide at E:T ratios of <0.2:1 are shown in FIG. 8. Exemplary data demonstrating that T cells stimulated to recognize a RAS^G12Vneoantigen on HLA-A11:01 kill SW620 cells that naturally have the RASG12V mutation and are transduced with HLA-A11:01 are shown in FIG. 9. Exemplary data demonstrating that T cells stimulated to recognize a RAS^G12Vneoantigen on HLA-A03:01 kill NCI-H441 cells that naturally have the RASG12V mutation and HLA-A03:01 are shown in FIG. 10. Exemplary data demonstrating that T cells stimulated to recognize a GATA3 neoantigen on HLA-A02:01 kill 293T cells that naturally have HLA-A02:01 and are transduced with the GATA3 neoORF are shown in FIGS. 22 and 23.

Example 5. Precision NEO-STIM Process Applied to Tissue-Specific Antigens

Antigens that are specifically expressed in a non-essential tissue can be targeted if a tumor arises in such a tissue. For example, antigens specifically expressed in prostate tissues can be targeted in the context of metastatic prostate cancer in which the primary tumor was resected, because the only cells expressing these antigens are metastatic cancer cells. There are multiple such non-essential tissues. As an example, prostate cells were evaluated using two methodologies to discover potential prostate-specific antigens. In one approach, prostate tissue or prostate cancer cell lines were evaluated using HLA-MS as outlined in Example 3. This approach can lead to identification of antigens that are validated to be processed and presented. Exemplary data from this approach is shown in FIG. 25A. In another approach, genes known to be expressed specifically in prostate cells can be evaluated through one or more MHC binding and presentation prediction software. A peptide-MHC prediction algorithm was generated and was used for these studies. As in Examples 2, 3 and 4, mass spectrometry, cellular and immunological assays further help validate a predicted peptide-HLA pair. Exemplary results from this analysis on 4 genes known to be specifically expressed in the prostate (KLK2, KLK3, KLK4, TGM4) are shown in the table below. These epitopes were further subjected to immunogenicity studies as in Example 2. The epitopes that are prefixed with ‘*’, were shown to induce positive CD8+ T cell response in either one or both the donors (marked as 1 or 2 in column 6 respectively) and also demonstrated in FIG. 25B.

TABLE 12

Predicted
RECON

SEQ ID

Affinity
Percent
Immunogenicity

Peptide
NO:
Allele
Gene
(nM)
Rank
(#donors/2)

SLQCVSLHL
1394
HLA-A02:01
KLK2
39.4
0.4

LVLSIALSV
1395
HLA-A02:01
KLK2
54.9
1.1

VILGVHLSV
1396
HLA-A02:01
KLK2
62.1
0.4

VLAPQESSV
1397
HLA-A02:01
KLK2
65.7
0.08

SLQCVSLHLL
1398
HLA-A02:01
KLK2
90.3
0.4

MLLRLSEPA
1399
HLA-A02:01
KLK2; KLK3
56
2.5

LTMPALPMV
1400
HLA-A02:01
KLK3
14.3
1.1

FLTLSVTWIA
1401
HLA-A02:01
KLK3
16.9
3.5

KLQCVDLHV
1402
HLA-A02:01
KLK3
21.2
0.3

*FLTPKKLQCV
1403
HLA-A02:01
KLK3
126.4
0.17
1

FLRPGDDSTL
1404
HLA-A02:01
KLK3
982.7
0.4

*FLGYLILGV
1405
HLA-A02:01
KLK4
6.3
0.05
1

*LLANDLMLI
1406
HLA-A02:01
KLK4
10.7
0.4
2

*FQNSYTIGL
1407
HLA-A02:01
KLK4
15.1
1.6
2

MLIKLDESV
1408
HLA-A02:01
KLK4
17.6
0.25

VLQCVNVSV
1409
HLA-A02:01
KLK4
19.2
0.1

*LLANGRMPTV
7
HLA-A02:01
KLK4
25.9
0.25
2

*ILNDTGCHYV
1410
HLA-A02:01
TGM4
17.2
0.1
1

*FQYPEFSIEL
1411
HLA-A02:01
TGM4
21.2
1
1

ILGKYQLNV
1412
HLA-A02:01
TGM4
22
0.3

LLGNSVNFTV
1413
HLA-A02:01
TGM4
27.8
0.7

*VLDCCISLL
1414
HLA-A02:01
TGM4
30.6
0.4
1

ILGSFELQL
1415
HLA-A02:01
TGM4
31.2
0.25

*RLIWLVKMV
1416
HLA-A02:01
TGM4
64.4
0.17
1

VLLGNSVNFTV
1417
HLA-A02:01
TGM4
83.7
0.6

TLAIPLTDV
1418
HLA-A02:01
TGM4
149.2
0.25

In a further assay, T cells that are specific for the peptides indicated in the table were tested for ability to kill target cells as described in Example 4. An exemplary data is presented in FIG. 25C, where KLK4 prostate specific epitope were co-cultured with 293T-GFP cells either loaded with 2 uM of peptide or not loaded. Peptide loaded target cells were killed to a much greater extent (right image) compared to the no peptide control (left image).

Example 6. Enrichment of Target Antigen Activated T Cells

Tumor antigen responsive T cells may be further enriched. In this example, multiple avenues for enrichment of antigen responsive T cells are explored and results presented. After the initial stimulation of antigen-specific T cells (Example 2, Steps 1-5), an enrichment procedure can be used prior to further expansion of these cells. As an example, stimulated cultures and pulsed with the same peptides used for the initial stimulation on day 13, and cells upregulating 4-1BB are enriched using Magnetic-Assisted Cell Separation (MACS; Miltenyi). These cells can then be further expanded, for example, using anti-CD3 and anti-CD28 microbeads and low-dose IL-2. As shown in FIG. 19A (middle row) and FIG. 19B (middle column), this approach can enrich for multiple antigen-specific T cell populations. As another example, T cells that are stained by multimers can be enriched by MACS on day 14 of stimulation and further expanded, for example, using anti-CD3 and anti-CD28 microbeads and low-dose IL-2. As shown in FIG. 19A (bottom row) and FIG. 19B (right column), this approach can enrich for multiple antigen-specific T cell populations.

Example 7. Immunogenicity Assays for Selected Peptides

After maturation of DCs, PBMCs (either bulk or enriched for T cells) are added to mature dendritic cells with proliferation cytokines. Cultures are monitored for peptide-specific T cells using a combination of functional assays and/or tetramer staining. Parallel immunogenicity assays with the modified and parent peptides allowed for comparisons of the relative efficiency with which the peptides expanded peptide-specific T cells. In some embodiments, the peptides elicit an immune response in the T cell culture comprises detecting an expression of a FAS ligand, granzyme, perforins, IFN, TNF, or a combination thereof in the T cell culture.

Immunogenicity can be measured by a tetramer assay. MHC tetramers are purchased or manufactured on-site, and are used to measure peptide-specific T cell expansion in the immunogenicity assays. For the assessment, tetramer is added to 1×10{circumflex over ( )}5 cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) according to manufacturer's instructions. Cells are incubated in the dark for 20 minutes at room temperature. Antibodies specific for T cell markers, such as CD8, are then added to a final concentration suggested by the manufacturer, and the cells are incubated in the dark at 4 degrees Celsius for 20 minutes. Cells are washed with cold FACS buffer and resuspended in buffer containing 1% formaldehyde. Cells are acquired on a FACS Calibur (Becton Dickinson) instrument, and are analyzed by use of Cellquest software (Becton Dickinson). For analysis of tetramer positive cells, the lymphocyte gate is taken from the forward and side-scatter plots. Data are reported as the percentage of cells that were CD8⁺/Tetramer⁺.

Immunogenicity can be measured by intracellular cytokine staining. In the absence of well-established tetramer staining to identify antigen-specific T cell populations, antigen-specificity can be estimated using assessment of cytokine production using well-established flow cytometry assays. Briefly, T cells are stimulated with the peptide of interest and compared to a control. After stimulation, production of cytokines by CD4+ T cells (e.g., IFNγ and TNFα) are assessed by intracellular staining. These cytokines, especially IFNγ, used to identify stimulated cells.

In some embodiments the immunogenicity is measured by measuring a protein or peptide expressed by the T cell, using ELISpot assay. Peptide-specific T cells are functionally enumerated using the ELISpot assay (BD Biosciences), which measures the release of IFNγ from T cells on a single cell basis. Target cells (T2 or HLA-A0201 transfected C1Rs) were pulsed with 10 μM peptide for one hour at 37 degrees C., and washed three times. 1×10{circumflex over ( )}5 peptide-pulsed targets are co-cultured in the ELISPOT plate wells with varying concentrations of T cells (5×10{circumflex over ( )}2 to 2×10{circumflex over ( )}3) taken from the immunogenicity culture. Plates are developed according to the manufacturer's protocol, and analyzed on an ELISPOT reader (Cellular Technology Ltd.) with accompanying software. Spots corresponding to the number of IFN gamma-producing T cells are reported as the absolute number of spots per number of T cells plated. T cells expanded on modified peptides are tested not only for their ability to recognize targets pulsed with the modified peptide, but also for their ability to recognize targets pulsed with the parent peptide.

CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide. The lytic granules of T cells have a lipid bilayer that contains lysosomal-associated membrane glycoproteins (“LAMPs”), which include the molecules CD107a and b. When cytotoxic T cells are activated through the T cell receptor, the membranes of these lytic granules mobilize and fuse with the plasma membrane of the T cell. The granule contents are released, and this leads to the death of the target cell. As the granule membrane fuses with the plasma membrane, C107a and b are exposed on the cell surface, and therefore are markers of degranulation. Because degranulation as measured by CD107a and b staining is reported on a single cell basis, the assay is used to functionally enumerate peptide-specific T cells. To perform the assay, peptide is added to HLA-A0201-transfected cells C1R to a final concentration of 20 μM, the cells were incubated for 1 hour at 37 degrees C., and washed three times. 1×10{circumflex over ( )}5 of the peptide-pulsed C1R cells were aliquoted into tubes, and antibodies specific for CD107a and b are added to a final concentration suggested by the manufacturer (Becton Dickinson). Antibodies are added prior to the addition of T cells in order to “capture” the CD107 molecules as they transiently appear on the surface during the course of the assay. 1×10{circumflex over ( )}5 T cells from the immunogenicity culture are added next, and the samples were incubated for 4 hours at 37 degrees C. The T cells are further stained for additional cell surface molecules such as CD8 and acquired on a FACS Calibur instrument (Becton Dickinson). Data is analyzed using the accompanying Cellquest software, and results were reported as the percentage of CD8+ CD107 a and b+ cells.

Cytotoxic activity is measured using a chromium release assay. Target T2 cells are labeled for 1 hour at 37 degrees C. with Na51Cr and washed 5×10{circumflex over ( )}3 target T2 cells were then added to varying numbers of T cells from the immunogenicity culture. Chromium release is measured in supernatant harvested after 4 hours of incubation at 37 degrees C. The percentage of specific lysis is calculated as:

Experimental release−spontaneous release/Total release−spontaneous release×100

Immunogenicity assays were carried out to assess whether each peptide can elicit a T cell response by antigen-specific expansion. Though current methods are imperfect, and therefore negative results do not imply a peptide is incapable of inducing a response, a positive result demonstrates that a peptide can induce a T cell response. Several peptides from Table 3 were tested for their capacity to elicit CD8+ T cell responses with multimer readouts as described. Each positive result was measured with a second multimer preparation to avoid any preparation biases. In an exemplary assay, HLA-A02:01+ T cells were co-cultured with monocyte-derived dendritic cells loaded with TMPRSS2::ERG fusion neoepitope (ALNSEALSV (SEQ ID NO: 992); HLA-A02:01) for 10 days. CD8+ T cells were analyzed for antigen-specificity for TMPRSS2::ERG fusion neoepitope using multimers (initial: BV421 and PE; validation: APC and BUV396).

While antigen-specific CD8+ T cell responses are readily assessed using well-established HLA Class I multimer technology, CD4+ T cell responses require a separate assay to evaluate because HLA Class II multimer technology is not well-established. In order to assess CD4+ T cell responses, T cells were re-stimulated with the peptide of interest and compared to a control. In the case of a completely novel sequence (e.g., arising from a frame-shift or fusion), the control was no peptide. In the case of a point-mutation, the control was the WT peptide. After stimulation, production of cytokines by CD4+ T cells (e.g., IFNγ and TNFα) were assessed by intracellular staining. These cytokines, especially IFNγ, used to identify stimulated cells. Antigen-specific CD4+ T cell responses showed increased cytokine production relative to control.

Example 8. Cell Expansion and Preparation

To prepare APCs, the following method was employed (a) obtain of autologous immune cells from the peripheral blood of the patient; enrich monocytes and dendritic cells in culture; load peptides and mature DCs.

T Cell Induction (Protocol 1)

First induction: (a) Obtaining autologous T cells from an apheresis bag; (b) Depleting CD25+ cells and CD14+ cells, alternatively, depleting only CD25+ cells; (c) Washing the peptide loaded and mature DC cells, resuspending in the T cell culture media; (d) Incubating T cells with the matured DC.

Second induction: (a) Washing T cells, and resuspending in T cell media, and optionally evaluating a small aliquot from the cell culture to determine the cell growth, comparative growth and induction of T cell subtypes and antigen specificity and monitoring loss of cell population; (b) Incubating T cells with mature DC.

Third induction: (a) Washing T cells, and resuspending in T cell media, and optionally evaluating a small aliquot from the cell culture to determine the cell growth, comparative growth and induction of T cell subtypes and antigen specificity and monitoring loss of cell population; (b) Incubating T cells with mature DC.

To harvest peptide activated t cells and cryopreserve the T cells, the following method was employed (a) Washing and resuspension of the final formulation comprising the activated T cells which are at an optimum cell number and proportion of cell types that constitutes the desired characteristics of the Drug Substance (DS). The release criteria testing include inter alia, Sterility, Endotoxin, Cell Phenotype, TNC Count, Viability, Cell Concentration, Potency; (b) Filling drug substance in suitable enclosed infusion bags; (c) Preservation until time of use.

Example 9. Methods of Functional Characterization of the CD4+ and CD8+ Neoantigen-Specific T Cells

Neoantigens, which arise in cancer cells from somatic mutations that alter protein-coding gene sequences, are emerging as an attractive target for immunotherapy. They are uniquely expressed on tumor cells as opposed to healthy tissue and may be recognized as foreign antigens by the immune system, increasing immunogenicity. T cell manufacturing processes were developed to raise memory and de novo CD4+ and CD8+ T cell responses to patient-specific neoantigens through multiple rounds of ex-vivo T cell stimulation, generating a neoantigen-reactive T cell product for use in adoptive cell therapy. Detailed characterization of the stimulated T cell product can be used to test the many potential variables these processes utilize.

To probe T cell functionality and/or specificity, an assay was developed to simultaneously detect antigen-specific T cell responses and characterize their magnitude and function. This assay employs the following steps. First T cell-APC co-cultures were used to elicit reactivity in antigen-specific T cells. Optionally, sample multiplexing using fluorescent cell barcoding is employed. To identify antigen-specific CD8+ T cells and to examine T cell functionality, staining of peptide-MHC multimers and multiparameter intracellular and/or cell surface cell marker staining were probed simultaneously using FACS analysis. The results of this streamlined assay demonstrated its application to study T cell responses induced from a healthy donor. Neoantigen-specific T cell responses induced toward peptides were identified in a healthy donor. The magnitude, specificity and functionality of the induced T cell responses were also compared. Briefly, different T cell samples were barcoded with different fluorescent dyes at different concentrations (see, e.g., Example 19). Each sample received a different concentration of fluorescent dye or combination of multiple dyes at different concentrations. Samples were resuspended in phosphate-buffered saline (PBS) and then fluorophores dissolved in DMSO (typically at 1:50 dilution) were added to a maximum final concentration of 5 μM After labeling for 5 min at 37° C., excess fluorescent dye was quenched by the addition of protein-containing medium (e.g. RPMI medium containing 10% pooled human type AB serum). Uniquely barcoded T cell cultures were challenged with autologous APC pulsed with the antigen peptides as described above.

The differentially labeled samples were combined into one FACS tube or well, and pelleted again if the resulting volume is greater than 100 μL. The combined, barcoded sample (typically 100 μL) was stained with surface marker antibodies including fluorochrome conjugated peptide-MHC multimers. After fixation and permeabilization, the sample was additionally stained intracellularly with antibodies targeting TNF-α and IFN-γ.

The cell marker profile and MEC tetramer staining of the combined, barcoded T cell sample were then analyzed simultaneously by flow cytometry on flow cytometer. Unlike other methods that analyze cell marker profiles and MEC tetramer staining of a T cell sample separately, the simultaneous analysis of the cell marker profile and MEC tetramer staining of a T cell sample described in this example provides information about the percentage of T cells that are both antigen specific and that have increased cell marker staining. Other methods that analyze cell marker profiles and MEC tetramer staining of a T cell sample, separately determine the percentage of T cells of a sample that are antigen specific, and separately determine the percentage of T cells that have increased cell marker staining, only allowing correlation of these frequencies.

The simultaneous analysis of the cell marker profile and MEC tetramer staining of a T cell sample described in this example does not rely on correlation of the frequency of antigen specific T cells and the frequency of T cells that have increased cell marker staining; rather, it provides a frequency of T cells that are both antigen specific and that have increased cell marker staining. The simultaneous analysis of the cell marker profile and MEC tetramer staining of a T cell sample described in this example allows for determination on a single cell level, those cells that are both antigen specific and that have increased cell marker staining.

To evaluate the success of a given induction process, a recall response assay was used followed by a multiplexed, multiparameter flow cytometry panel analysis. A sample taken from an induction culture was labeled with a unique two-color fluorescent cell barcode. The labeled cells were incubated on antigen-loaded DCs or unloaded DCs overnight to stimulate a functional response in the antigen-specific cells. The next day, uniquely labeled cells were combined prior to antibody and multimer staining according to Table 9 below.

TABLE 9

Marker
Fluorochrome
Purpose

CD19/CD16/CD14
BUV395
Cell exclusion

Live/Dead
Near-IR
Dead cell exclusion

CD3
BUV805
Lineage gating

CD4
Alexa Fluor 700
Lineage gating

CD8
PerCP-Cy5.5
Lineage gating

Barcode 1
CFSE
Sample multiplexing

Barcode 2
TagIT Violet
Sample multiplexing

Multimer 1
PE
CD8+ antigen specificity

Multimer 2
BV650
CD8+ antigen specificity

IFNγ
APC
Functionality

TNFα
BV711
Functionality

CD107a
BV786
Cytotoxicity

4-1BB
PE/Dazzle 594
Activation

Patient-specific neoantigens were predicted using bioinformatics engine. Synthetic long peptides covering the predicted neoantigens were used as immunogens in the stimulation protocol to assess the immunogenic capacity. The stimulation protocol involves feeding these neoantigen-encoding peptides to patient-derived APCs, which are then co-cultured with patient-derived T cells to prime neoantigen specific T cells.

Multiple rounds of stimulations are incorporated in the stimulation protocol to prime, activate and expand memory and de novo T cell responses. The specificity, phenotype and functionality of these neoantigen-specific T cells was analyzed by characterizing these responses with the following assays: Combinatorial coding analysis using pMHC multimers was used to detect multiple neoantigen-specific CD8+ T cell responses. A recall response assay using multiplexed, multiparameter flow cytometry was used to identify and validate CD4+ T cell responses. The functionality of CD8+ and CD4+ T cell responses was assessed by measuring production of pro-inflammatory cytokines including IFN-γ and TNFα, and upregulation of the CD107a as a marker of degranulation. A cytotoxicity assay using neoantigen-expressing tumor lines was used to understand the ability of CD8+ T cell responses to recognize and kill target cells in response to naturally processed and presented antigen. The cytotoxicity was measured by the cell surface upregulation of CD107a on the T cells and upregulation of active Caspase3 on neoantigen-expressing tumor cells. The stimulation protocol was successful in the expansion of pre-existing CD8+ T cell responses, as well as the induction of de novo CD8+ T cell responses (Table 10).

TABLE 10

(“DEAH” disclosed as SEQ ID NO: 1419)

HUGO

Patient
Symbol
Full Gene Name
Type

NV10
SRSF1E>K
Serine and Arginine Rich Splicing Factor 1
CD8

ARAP1Y>H
Ankyrin Repeat And PH Domain

PKDREJG>R
Polycystin Family Receptor For Egg Jelly

MKRN1_S>L
Makorin Ring Finger Protein 1
CD4

CREBBP_S>L
CRREB Binding Protein

TPCN1_K>E
Two Pore Segment Channel 1

NV6
AASDH_neoORF
Aminoadipate-Semialdehyde Dehydrogenase
CD8

ACTN4_K>N
Actinin Alpha 4

CSNK1A1_S>L
Casein Kinase 1 Alpha 1

DHX40_neoORF
DEAH-Box Helicase 40

GLI3_P>L
GLI Family Zinc Finger 3

QARS_R>W
Glutamyl-tRNA Synthetase

FAM178B_P>L
Family With Sequence Similarity 178 Member 8

RPS26_P>L
Ribosomal Protein S26

Using PBMCs from a melanoma patient a clinical study performed by Applicant's group, expansion of a pre-existing CD8+ T cell response was observed from 4.5% of CD8+ T cells to 72.1% of CD8+ T cells (SRSF1E_>K). Moreover, the stimulation protocol was effective in inducing two presumed de novo CD8+ T cell responses towards patient-specific neoantigens (exemplary de novo CD8+ T cell responses: ARAP1_Y>H: 6.5% of CD8+ T cells and PKDREJ_G>R: 13.4% of CD8+ T cells; no cells were detectable prior to the stimulation process). The stimulation protocol successfully induced seven de novo CD8+ T cell responses towards both previously described and novel model neoantigens using PBMCs from another melanoma patient, NV6, up to varying magnitudes (ACTN4_K>NCSNK1A1_S>L, DHX40neoORF 7, GLI3_P>L, QARS_R>W, FAM178B_P>L, and RPS26_P>L, range: 0.2% of CD8+ T cells up to 52% of CD8+ T cells). Additionally, a CD8+ memory T cell response towards a patient-specific neoantigen was expanded (AASDHneoORF, up to 13% of CD8+ T cells post stimulation).

The induced CD8+ T cells from the patient was characterized in more detail. Upon re-challenge with mutant peptide loaded DCs, neoantigen-specific CD8+ T cells exhibited one, two and/or all three functions (16.9% and 65.5% functional CD8+ pMHC+ T cells for SRSF1E>K and ARAP1Y>H, respectively. When re-challenged with different concentrations of neoantigen peptides, the induced CD8+ T cells responded significantly to mutant neoantigen peptide but not to the wildtype peptide. In said patient, CD4+ T cell responses were identified using a recall response assay with mutant neoantigen loaded DCs. Three CD4+ T cell responses were identified (MKRN1S>L, CREBBPS>L and TPCN1K>E) based on the reactivity to DCs loaded with mutant neoantigen peptide. These CD4+ T cell responses also showed a polyfunctional profile when re-challenged with mutant neoantigen peptide. 31.3%, 34.5% & 41.9% of CD4+ T cells exhibited one, two and/or three functions; MKRN1S>L, CREBBPS>L and TPCN1K>E responses, respectively.

The cytotoxic capacity of the induced CD8+ responses from said patient was also assessed. Both SRSF1E>K and ARAP1Y>H responses showed a significant upregulation of CD107a on the CD8+ T cells and active Caspase3 on the tumor cells transduced with the mutant construct after co-culture.

Using the stimulation protocol, predicted patient-specific neoantigens, as well as model neoantigens, were confirmed to be immunogenic by the induction of multiple neoantigen-specific CD8+ and CD4+ T cell responses in patient material. The ability to induce polyfunctional and mutant-specific CD8+ and CD4+ T cell responses proves the capability of predicting high-quality neoantigens and generating potent T cell responses. The presence of multiple enriched neoantigen-specific T cell populations (memory and de novo) at the end of the stimulation process demonstrates the ability to raise new T cell responses and generate effective cancer immunotherapies to treat cancer patients.

Exemplary materials for T cell culture are provided below: Materials: AIM V media (Invitrogen)Human FLT3L; preclinical CellGenix #1415-050 Stock 50 ng/μL TNFα; preclinical CellGenix #1406-050 Stock 10 ng/μL; IL-1β, preclinical CellGenix #1411-050 Stock 10 ng/μL; PGE1 or Alprostadil—Cayman from Czech republic Stock 0.5 μg/μL; R10 media—RPMI 1640 glutamax+10% Human serum+1% PenStrep; 20/80 Media—18% AIM V+72% RPMI 1640 glutamax+10% Human Serum+1% PenStrep; IL7 Stock 5 ng/μL; IL15 Stock 5 ng/μL; DC media (Cellgenix); CD14 microbeads, human, Miltenyi #130-050-201, Cytokines and/or growth factors, T cell media (AIM V+RPMI 1640 glutamax+serum+PenStrep), Peptide stocks—1 mM per peptide (HIV A02—5-10 peptides, HIV B07—5-10 peptides, DOM—4-8 peptides, PIN—6-12 peptides).

COMPOSITIONS AND METHODS FOR PREPARING T CELL COMPOSITIONS AND USES THEREOF

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