Protein antigens and uses thereof

Abstract

To provide non-mutated protein epitopes expressed in cancer cells, useful alone or in combination with other tumor-associated peptides, anti-cancer agents, or immunomodulatory agents for treating cancer.SOLUTION: Provided herein is an isolated antigenic peptide comprising an epitope from a sequence in Tables 1 to 6. The present disclosure is also directed to an isolated antigenic peptide that is 100 amino acids or less in length and comprises an epitope from a sequence in Tables 1 to 6. In one embodiment, the isolated antigenic peptide is a retroviral antigen. In another embodiment, the isolated antigenic peptide is a non-mutated overexpressed antigen. In another embodiment, the isolated antigenic peptide is a viral antigen.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference This application claims the benefit of priority of U.S. Provisional Application No. 62 / 480,593, filed Apr. 3, 2017; U.S. Provisional Application No. 62 / 480,596, filed Apr. 3, 2017; and U.S. Provisional Application No. 62 / 480,597, filed Apr. 3, 2017 (each of which is hereby incorporated by reference in its entirety).

[0002] Field The field of the invention relates to immunotherapeutic peptides, nucleic acids encoding the peptides, peptide binders, and their use, for example, in the immunotherapy of cancer. In one aspect, the invention provides non-mutated protein epitopes expressed in cancer cells that are useful alone or in combination with other tumor-associated peptides, anti-cancer agents, or immunomodulatory agents for treating cancer.

Background Art

[0003] Background Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (such as adjuvants, cytokines or TLR ligands) that act together to induce antigen-specific cytotoxic T lymphocytes (CTLs) that recognize and lyse tumor cells. Such vaccines contain either a shared tissue restricted tumor antigen or a mixture of common and patient-specific antigens in the form of a whole tumor cell preparation. Shared tissue restricted tumor antigens are ideally immunogenic proteins that have selective expression in tumors across many individuals and are generally delivered to patients as synthetic peptides or recombinant proteins. In contrast, whole tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations or whole mRNAs. Since whole tumor cells are isolated from the patient themselves, the cells may contain patient-specific tumor antigens as well as common tumor antigens. Finally, there are neoantigens, a third class of tumor antigens, consisting of proteins that have tumor-specific mutations (which may be patient-specific or common) that result in changes in amino acid sequence. Therefore, there is still a need to develop additional cancer therapeutics.

Summary of the Invention

Means for Solving the Problems

[0004] Summary Disclosed herein are isolated antigenic peptides comprising epitopes derived from the sequences in Table 1 or Table 2. The disclosure also relates to isolated antigenic peptides of 100 amino acids in length or less comprising epitopes derived from the sequences in Table 1 or Table 2. The disclosure also relates to isolated antigenic peptides comprising epitopes derived from the sequences in Table 3 or Table 4. The disclosure also relates to isolated antigenic peptides of 100 amino acids in length or less comprising epitopes derived from the sequences in Table 3 or Table 4. The disclosure also relates to isolated antigenic peptides comprising epitopes derived from the sequences in Table 5 or Table 6. The disclosure also relates to isolated antigenic peptides of 100 amino acids in length or less comprising epitopes derived from the sequences in Table 5 or Table 6.

[0005] In one embodiment, the isolated antigenic peptide is a retroviral antigen. In another embodiment, the isolated antigenic peptide is a non-mutated overexpressed antigen. In another embodiment, the isolated antigenic peptide is a viral antigen.

[0006] In one embodiment, the isolated antigenic peptide is between about 5 and about 50 amino acids in length. In another embodiment, the isolated antigenic peptide is between about 15 and about 35 amino acids in length. In another embodiment, the isolated antigenic peptide is about 15 amino acids in length or less. In another embodiment, the isolated antigenic peptide is between about 8 and about 11 amino acids in length. In another embodiment, the isolated antigenic peptide is 9 or 10 amino acids in length. In one embodiment, the isolated antigenic peptide binds to major histocompatibility complex (MHC) class I. In another embodiment, the isolated antigenic peptide binds to MHC class I with a binding affinity of less than about 500 nM.

[0007] In one embodiment, the isolated antigenic peptide is about 30 amino acids in length or less. In another embodiment, the isolated antigenic peptide is between about 6 and about 25 amino acids in length. In another embodiment, the isolated antigenic peptide is between about 15 and about 24 amino acids in length. In another embodiment, the isolated antigenic peptide is between about 9 and about 15 amino acids in length. In one embodiment, the isolated antigenic peptide binds to MHC class II. In another embodiment, the isolated antigenic peptide binds to MHC class II with a binding affinity of less than about 1000 nM.

[0008] In one embodiment, the isolated antigenic peptide further comprises adjacent amino acids. In another embodiment, the adjacent amino acids are not the native adjacent amino acids. In one embodiment, the isolated antigenic peptide is linked to at least a second antigenic peptide. In another embodiment, the peptides are linked using a polyglycine or polyserine linker. In another embodiment, the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM. In another embodiment, the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 500 nM. In another embodiment, both epitopes bind to human leukocyte antigen (HLA)-A, -B, -C, -DP, -DQ, or -DR. In another embodiment, the isolated antigenic peptide binds to class I HLA and the second antigenic peptide binds to class II HLA. In another embodiment, the isolated antigenic peptide binds to class II HLA and the second antigenic peptide binds to class I HLA.

[0009] In one embodiment, the isolated antigenic peptide further comprises a modification that increases in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. In another embodiment, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation, HESylation, recombinant PEG mimetic, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of an amino acid mimic, or addition of a non-natural amino acid. In one embodiment, the cell to be targeted is an antigen presenting cell. In another embodiment, the antigen presenting cell is a dendritic cell. In another embodiment, the dendritic cell is targeted using the DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, or CD1a marker. In another embodiment, the dendritic cell is targeted using the CD141, DEC205, or XCR1 marker.

[0010] In one embodiment, an in vivo delivery system comprising the isolated antigenic peptide described herein is provided herein. In another embodiment, the delivery system comprises a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, or liposomes. In another embodiment, the cell-penetrating peptide is the TAT peptide, herpes simplex virus VP22, transportan, or Antp.

[0011] In one embodiment, a cell comprising the isolated antigenic peptide described herein is provided herein. In another embodiment, the cell is an antigen presenting cell. In another embodiment, the cell is a dendritic cell.

[0012] In one embodiment, a composition comprising the isolated antigenic peptides described herein is provided herein. In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides comprising tumor-specific epitopes defined in Table 1 or Table 2. In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides comprising tumor-specific epitopes defined in Table 3 or Table 4. In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides comprising tumor-specific epitopes defined in Table 5 or Table 6.In another embodiment, it further comprises between 2 and 20 antigenic peptides. In another embodiment, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 additional antigenic peptides. In another embodiment, the composition comprises between about 4 and about 20 additional antigenic peptides. In another embodiment, the additional antigenic peptides are specific to the tumor of an individual patient. In another embodiment, the antigenic peptides are selected by identifying the expression differences between the transcriptome or proteome of a patient's tumor sample and the transcriptome or proteome of a non-tumor sample. In another embodiment, the sample is fresh or formalin-fixed paraffin-embedded tumor tissue, freshly isolated cells, or circulating tumor cells. In some embodiments, the sequence of the antigenic peptide is determined by next-generation sequencing.

[0013] In one embodiment, an isolated polynucleotide encoding the isolated neoantigenic peptide described herein is provided herein. In another embodiment, the isolated polynucleotide is RNA, optionally self-amplifying RNA. In another embodiment, the RNA is modified to increase stability, increase cell targeting, increase translation efficiency, adjuvant activity, cytosolic accessibility, and / or decrease cytotoxicity. In another embodiment, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC content, incorporation of modified nucleosides, incorporation of a 5'-cap or cap analog, and / or incorporation of an unmasked poly-A sequence.

[0014] In one embodiment, a cell comprising the polynucleotide described herein is provided herein.

[0015] In one embodiment, a vector comprising the polynucleotide described herein is provided herein. In another embodiment, the polynucleotide is operably linked to a promoter. In another embodiment, the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus or virion. In another embodiment, the vector is an adeno-associated virus, herpes virus, lentivirus or pseudotype thereof.

[0016] In one embodiment, an in vivo delivery system comprising the isolated polynucleotide described herein is provided herein. In another embodiment, the delivery system comprises spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids or nanoparticles.

[0017] In one embodiment, a cell comprising the vector or delivery system described herein is provided herein. In another embodiment, the cell is an antigen-presenting cell. In another embodiment, the cell is a dendritic cell. In another embodiment, the cell is an immature dendritic cell.

[0018] In one embodiment, a composition comprising at least one polynucleotide described herein is provided herein. In another embodiment, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated polynucleotides. In another embodiment, the composition comprises between about 2 and about 20 polynucleotides. In another embodiment, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 additional antigenic polynucleotides encoding additional antigenic peptides. In another embodiment, the composition comprises between about 4 and about 20 additional antigenic polynucleotides. In another embodiment, the isolated polynucleotide and the additional antigenic polynucleotide are linked. In another embodiment, the polynucleotides are linked using a nucleic acid encoding a polyglycine or polyserine linker. In another embodiment, at least one additional antigenic peptide is specific for the tumor of an individual patient. In another embodiment, the antigenic peptide is selected by identifying the differential expression between the transcriptome or proteome of a patient's tumor sample and the transcriptome or proteome of a non-tumor sample.In another embodiment, the sample is fresh or formalin-fixed paraffin-embedded tumor tissue, freshly isolated cells, or circulating tumor cells. In some embodiments, the sequence of the antigenic peptide is determined by next-generation sequencing.

[0019] In one embodiment, a T cell receptor (TCR) capable of binding to at least one antigenic peptide described herein is provided herein. In another embodiment, the TCR can bind to an antigenic peptide isolated in the context of MHC class I or class II.

[0020] In one embodiment, a chimeric antigen receptor is provided herein that comprises (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition portion capable of binding to an isolated antigenic peptide described herein. In another embodiment, CD3 zeta is the T cell activation molecule. In another embodiment, the chimeric antigen receptor further comprises at least one co-stimulatory signaling domain. In another embodiment, the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI gamma. In another embodiment, the antigen recognition portion can bind to an antigenic peptide isolated in the context of MHC class I or class II. In another embodiment, the chimeric antigen receptor comprises a CD3 zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region. In another embodiment, the tumor-specific epitope is located in the extracellular domain of a tumor-associated polypeptide.

[0021] In one embodiment, a T cell comprising a T cell receptor or chimeric antigen receptor described herein is provided herein. In one embodiment, the T cell is a helper T cell or a cytotoxic T cell.

[0022] In one embodiment, a nucleic acid is provided herein that includes a promoter operably linked to a polynucleotide encoding a T cell receptor described herein. In another embodiment, the TCR can bind to at least one antigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In one embodiment, the nucleic acid includes a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor described herein. In another embodiment, the antigen recognition portion can bind to at least one antigenic peptide in the context of major histocompatibility complex (MHC) class I or class II. In another embodiment, the tumor-specific epitope is located in the extracellular domain of a tumor-associated polypeptide. In another embodiment, the nucleic acid includes a transmembrane region of CD3 zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1.

[0023] In one embodiment, an antibody is provided herein that can bind to at least one antigenic peptide listed in Table 1 or Table 2. In another embodiment, an antibody is provided herein that can bind to at least one antigenic peptide listed in Table 3 or Table 4. In another embodiment, an antibody is provided herein that can bind to at least one antigenic peptide listed in Table 5 or Table 6. In another embodiment, at least one antigenic peptide listed in Table 1 or Table 2 is a retroviral antigenic peptide. In another embodiment, at least one antigenic peptide listed in Table 3 or Table 4 is a non-mutated overexpressed antigenic peptide. In another embodiment, at least one antigenic peptide listed in Table 5 or Table 6 is a viral antigenic peptide.

[0024] In one embodiment, a modified cell transfected or transduced with a nucleic acid described herein is provided herein. In one embodiment, the modified cell is a T cell, tumor-infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4+ T cell, CD8+ T cell, or NK cell.

[0025] In one embodiment, a composition comprising a T cell receptor or chimeric antigen receptor described herein is provided herein. In another embodiment, the composition comprises autologous patient T cells containing a T cell receptor or chimeric antigen receptor described herein. In another embodiment, the composition further comprises an immune checkpoint inhibitor. In another embodiment, the composition further comprises at least two immune checkpoint inhibitors. In another embodiment, the immune checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. In another embodiment, the immune checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

[0026] In one embodiment, the composition further comprises an immunomodulator or an adjuvant. In another embodiment, the immunomodulator is a costimulatory ligand, TNF ligand, Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB. In another embodiment, the immunomodulator is at least one cancer cell or cancer cell extract. In another embodiment, the cancer cell is autologous to the subject in need of the composition. In another embodiment, the cancer cell is lysed or exposed to UV irradiation. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the adjuvant is selected from the group consisting of poly(I:C), polyICLC, STING agonist, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride, and QS21 stimulin. In another embodiment, the adjuvant induces humoral ) when administered to the subject. In another embodiment, the adjuvant induces type 1 helper T cells when administered to the subject.

[0027] In one embodiment, provided herein is a method of inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 1 or Table 2, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell of the present invention. In another embodiment, provided herein is a method of inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 3 or Table 4, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell of the present invention. In another embodiment, provided herein is a method of inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 5 or Table 6, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell of the present invention.

[0028] In one embodiment, provided herein is a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof, the method comprising administering to the subject a peptide, polynucleotide, vector, composition, antibody, or cell described herein. In one embodiment, the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, lung squamous cell carcinoma, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer. In one embodiment, the cancer is selected from the group consisting of melanoma, lung squamous cell carcinoma, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC. In one embodiment, the cancer is selected from the group consisting of cervical cancer, head and neck cancer, anal cancer, gastric cancer, Burkitt lymphoma, and nasopharyngeal cancer.

[0029] In one embodiment, the subject is a human. In another embodiment, the subject has cancer. In another embodiment, the cancer is selected from the group consisting of genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, triple-negative breast cancer (TNBC), smoldering multiple myeloma (SMM), Merkel cell carcinoma or osteosarcoma, hematological neoplasm, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-insensitive prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, cholangiocarcinoma, head and neck squamous cell cancer, soft tissue sarcoma, and small cell lung cancer. In another embodiment, the subject has undergone surgical removal of the tumor. In another embodiment, the peptide, polynucleotide, vector, composition or cell is administered via intravenous, intraperitoneal, intratumoral, intradermal or subcutaneous administration. In another embodiment, the peptide, polynucleotide, vector, composition or cell is administered to an anatomical site that drains into a lymph node basin. In another embodiment, the administration is made to multiple lymph node basins. In another embodiment, the administration is by subcutaneous or intradermal route. In another embodiment, the subject has undergone surgical removal of the tumor. In another embodiment, the peptide, polynucleotide, vector, composition or cell is administered via intravenous, intraperitoneal, intratumoral, intradermal or subcutaneous administration. In another embodiment, the peptide, polynucleotide, vector, composition or cell is administered to an anatomical site that drains into a lymph node basin. In another embodiment, the administration is made to multiple lymph node basins. In another embodiment, the administration is by subcutaneous or intradermal route.

[0030] In one embodiment of the method, a peptide is administered. In another embodiment, the administration is intratumoral. In another embodiment of the method, a polynucleotide, optionally RNA, is administered. In another embodiment, the polynucleotide is administered intravenously. In one embodiment of the method, cells are administered. In another embodiment, the cells are T cells or dendritic cells. In another embodiment, the peptide or polynucleotide comprises an antigen-presenting cell targeting moiety.

[0031] One embodiment of the method further comprises administering at least one immune checkpoint inhibitor to a subject. In another embodiment, the checkpoint inhibitor is a biologic or a small molecule. In another embodiment, the checkpoint inhibitor is selected from the group consisting of monoclonal antibodies, humanized antibodies, fully human antibodies, and fusion proteins or combinations thereof. In another embodiment, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. In another embodiment, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof. In another embodiment, two or more checkpoint inhibitors are administered. In another embodiment, the checkpoint inhibitors are: (i) ipilimumab or tremelimumab, and (ii) nivolumab. In another embodiment, the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order. In another embodiment, a peptide, polynucleotide, vector, composition, or cell is administered prior to the checkpoint inhibitor. In another embodiment, a peptide, polynucleotide, vector, composition, or cell is administered after the checkpoint inhibitor. In another embodiment, the administration of the checkpoint inhibitor is continued throughout antigen peptide, polynucleotide, vector, composition, or cell therapy. In another embodiment, the antigen peptide, polynucleotide, vector, composition, or cell therapy is administered to a subject who responds only partially or not at all to checkpoint inhibitor therapy. In another embodiment, the composition is administered intravenously or subcutaneously.In another embodiment, the checkpoint inhibitor is administered intravenously or subcutaneously. In another embodiment, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the administration site of the composition. In another embodiment, the composition is administered to the lymph node in the same inflow region as the checkpoint inhibitor.

[0032] In one embodiment of the method, an additional agent is administered. In another embodiment, the agent is a chemotherapeutic agent, an immunomodulatory agent, an immunometabolic modifier, a targeted therapy, radiation, an anti-angiogenic agent, or an agent that reduces immunosuppression. In another embodiment, the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an antimetabolite, or an anti-mitotic agent. In another embodiment, the additional agent is an anti-glucocorticoid-induced tumor necrosis factor family receptor (GITR) agonist antibody or antibody fragment, ibrutinib, docetaxel, cisplatin, or cyclophosphamide. In another embodiment, the administration induces a CD4 + T cell immune response. In another embodiment, the administration induces a CD4 + T cell immune response and a CD8 + T cell immune response.

[0033] In one embodiment, provided herein is a method for stimulating an immune response in a subject, comprising administering an effective amount of the modified cells or composition described herein. In another embodiment, the immune response is a cytotoxic and / or humoral immune response. In another embodiment, the method stimulates a T cell-mediated immune response in the subject. In another embodiment, the T cell-mediated immune response is directed against target cells. In another embodiment, the target cells are tumor cells. In another embodiment, the modified cells are transfected or transduced in vivo. In another embodiment, the modified cells are transfected or transduced ex vivo. In another embodiment, the modified cells are autologous patient T cells. In another embodiment, the autologous patient T cells are obtained from a patient who has received an antigenic peptide or nucleic acid vaccine. In another embodiment, the antigenic peptide or nucleic acid vaccine comprises at least one individualized antigen. In another embodiment, the antigenic peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 1 or 2. In another embodiment, the antigenic peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 3 or Table 4. In another embodiment, the antigenic peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 5 or Table 6. In another embodiment, at least one additional antigenic peptide listed in Table 1 or 2 is a retroviral antigenic peptide. In another embodiment, at least one additional antigenic peptide listed in Table 3 or Table 4 is a non-mutated overexpressed antigenic peptide. In another embodiment, at least one additional antigenic peptide listed in Table 5 or Table 6 is a viral antigenic peptide. In another embodiment, the patient has received a chemotherapeutic agent, an immunomodulatory agent, an immunometabolic modifying agent, a targeted therapy, or radiation prior to and / or during receiving the antigenic peptide or nucleic acid vaccine. In another embodiment, the patient receives treatment with at least one checkpoint inhibitor. In another embodiment, the autologous T cells are obtained from a patient who has already received at least one round of T cell therapy comprising an antigen. In another embodiment, the method further comprises adoptive T cell therapy.In another embodiment, the adoptive T cell therapy comprises autologous T cells. In another embodiment, the autologous T cells are targeted against a tumor antigen. In another embodiment, the adoptive T cell therapy further comprises allogeneic T cells. In another embodiment, the allogeneic T cells are targeted against a tumor antigen. In another embodiment, the adoptive T cell therapy is administered prior to the checkpoint inhibitor.

[0034] In one embodiment, a method for assessing the efficacy of a treatment comprises: (i) measuring the number or concentration of target cells in a first sample obtained from the subject prior to administration of modified cells; (ii) measuring the number or concentration of target cells in a second sample obtained from the subject after administration of modified cells. (iii) determining the number or concentration of target cells in the first sample; Provided herein is a method comprising determining an increase or decrease in the number or concentration of target cells in the second sample compared to the concentration of the first sample. In another embodiment, the treatment efficacy is determined by monitoring a clinical outcome; an increase, enhancement or prolongation of anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment; B cell activity; CD4 T cell activity, or a combination thereof. In another embodiment, the treatment efficacy is determined by monitoring a biomarker. In another embodiment, the biomarker is selected from the group consisting of CEA, Her-2 / neu, bladder tumor antigen, thyroglobulin, alpha fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, and b-type natriuretic peptide. In another embodiment, the clinical outcome is selected from the group consisting of tumor regression, tumor shrinkage, tumor necrosis, anti-tumor response by the immune system, tumor enlargement, recurrence or spread, or a combination thereof. In another embodiment, the treatment effect is predicted by the presence of T cells, or by the presence of a gene signature indicative of T cell inflammation, or a combination thereof.

[0035] In one embodiment, there is provided a method of treating cancer or initiating, enhancing or prolonging an anti-tumor response in a subject in need thereof, the method comprising administering to the subject (a) a peptide, polynucleotide, vector, composition, antibody or cell described herein, and (b) at least one checkpoint inhibitor. In another embodiment, the method further comprises administering an immunomodulatory factor or an adjuvant. In another embodiment, the immunomodulatory factor or adjuvant is selected from the group consisting of poly(I:C), polyICLC, STING agonist, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride and QS21 Stimulon, costimulatory ligands, TNF ligands, Ig superfamily ligands, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69 or 4-1BB. In another embodiment, the immunomodulatory factor or adjuvant is polyICLC. In another embodiment, the checkpoint inhibitor is an anti-PDl antibody or antibody fragment. In another embodiment, the inhibitor of the PD-1 pathway is nivolumab. In another embodiment, the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment.In another embodiment, the anti-CTLA4 antibody is ipilimumab or tremelimumab. In another embodiment, the method comprises administering both an anti-PD1 antibody and an anti-CTLA4 antibody. In another embodiment, the administration of the checkpoint inhibitor is initiated prior to the initiation of the administration of the peptide, polynucleotide, vector, composition, antibody or cell. In another embodiment, the administration of the checkpoint inhibitor is initiated after the initiation of the administration of the peptide, polynucleotide, vector, composition, antibody or cell. In another embodiment, the administration of the checkpoint inhibitor is initiated simultaneously with the initiation of the administration of the peptide, polynucleotide, vector, composition, antibody or cell. In another embodiment, the peptide, polynucleotide, vector, composition, antibody or cell is administered intravenously or subcutaneously. In another embodiment, the checkpoint inhibitor is administered intravenously or subcutaneously. In another embodiment, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the administration site of the peptide, polynucleotide, vector, composition, antibody or cell. In another embodiment, the peptide, polynucleotide, vector, composition, antibody or cell is administered to the same draining regional lymph node as the checkpoint inhibitor.

[0036] In one embodiment of the treatment method, the additional therapeutic agent is, for example, a chemotherapeutic agent or a biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer can be administered. Examples of chemotherapeutic agents or biotherapeutic agents include, but are not limited to, angiogenesis inhibitors such as hydroxyangiostatin K 1-3, DL-a-difluoromethylornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and thalidomide; DNA intercalating agents / crosslinkers such as bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammine platinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors such as (±)-amethopterin (methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, and mitomycin C; DNA-RNA transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin; S(-+-)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, histipidin, 2-imino-1-imidazolidineacetic acid (2-Immo-1-imidazoli-dineacetic acid; cyclocreatine), mevinolin, trico Enzyme inhibitors such as statin A, tilostatin AG34, and tilostatin AG879; gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, colecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, all-trans retinal (vitamin A aldehyde), all-trans retinoic acid (vitamin A acid), 9-cis retinoic acid, 13-cis retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone; microtubule inhibitors such as colchicine, docetaxel, dostarlimus 15, nocodazole, paclitaxel, podophyllotoxin, lysocine, vinblastine, vincristine, vindesine, and vinorelbine (navelbine); and unclassified therapeutic agents such as 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene diphosphonic acid, leuprorelin (leuprolide), luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, thapsigargin, and urinary trypsin inhibitor fragment (vikunin). Therapeutic agents include altretamine, amifostine, asparaginase, capecitabine, cladribine, cisapride, cyiarahirse, dacarbazine ( DT1C), dactinomycin, dronabinol, epoetin alpha, filgrastim ("filgrastim), fludarabine, gemcitabine, granisetron, ifosfamide, It may be irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, metoclopramide, mitotane, omeprazole, ondansetron, pilocarpine, prochlorperazine or topotecan hydrochloride. The therapeutic agent may be a monoclonal antibody such as rituximab (Rituxan®), alemtuzumab (Campath®), bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix®) and trastuzumab (Herceptin®), vemurafenib (Zelboraf®), imatinib mesylate (Gleevec®), erlotinib (Tarceva®), gefitinib (Iressa®), vismodegib (Erivedge™), 90Y-ibritumomab tiuxetan, 131I-tositumomab, ado-trastuzumab emtansine, lapatinib (Tykerb®), pertuzumab (Perjeta™), ado-trastuzumab emtansine (adcyla™), regorafenib (Stivarga®), sunitinib (Sutent®), denosumab (Xgeva®), sorafenib (Nexavar®), pazopanib (Votrient®), axitinib (Inlyta®), dasatinib (Sprycel®), nilotinib (Tasigna®), bosutinib (Bosulif®), ofatumumab (Arzerra®), obinutuzumab (Gazyva™), ibrutinib (Imbruvica™), idelalisib (Zydelig®), crizotinib (Xalkori®), erlotinib (Tarceva®), afatinib dimaleate (afatimbdimaleate)(Giiotrif (registered trademark)), ceritinib (LDK378 / Zykadia), tositumomab and 131I-tositumomab (Bexxar (registered trademark)), ibritumomab tiuxetan (Zevalin (registered trademark)), brentuximab vedotin (Adcetris (registered trademark)), bortezomib (Velcade (registered trademark)), siltuximab (Sylvant (trademark)), trametinib (ekinist (registered trademark)), dabrafenib (Tafmlar (registered trademark)), pembrolizumab (pembrolizimiab) (Keytruda (registered trademark)), carfilzomib (Kyprolis (registered trademark)), ramucirumab (Cyramza (trademark)), cabozantinib (Cometriq (trademark)), vandetanib (Caprelsa (registered trademark)) may be, and if necessary, the therapeutic agent is a neoantigen. The therapeutic agent may be a cytokine such as interferon (INF), interleukin (interlcukins) (IL) or hematopoietic growth factor. The therapeutic agent may be INF-α, IL-2, aldesleukin, IL-2, erythropoietin, granulocyte macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor. The therapeutic agent may be tamoxifen (Fareston (registered trademark)), fulvestrant (Faslodex (registered trademark)), anastrozole (Arimidex (registered trademark)), exemestane (Aromasin (registered trademark)), letrozole (Femara (registered trademark)), ziv-aflibercept (Zaltrap (registered trademark)), alitretinoin (Aiitretinoin) (Panretin (registered trademark)), temsirolimus (Torisel (registered trademark)), tretinoin (Vesanoid (registered trademark)), denileukin diftitox (Ontak (registered trademark)), vorinostat (Zoiinza (registered trademark)), romidepsin (Istodax (registered trademark)), bexarotene (Targretin (registered trademark)), pralatrexate (Foiotyn (registered trademark)), lenalidomide (lenaliomide) (Revlimid (registered trademark)), belinostat (Beleod aq (trademark), lenalidomide (Revlimid®), pomalidomide (Pomalyst®), cabazitaxel (Jevtana®), enzalutamide (Xtandi®), abiraterone acetate (Zytiga®), radium 223 chloride (Xofigo®) or everolimus (Afiniior®) and the like may be targeted therapies. Additionally, the therapeutic agent may be an epigenetic targeting agent such as an HDAC inhibitor, a kinase inhibitor, a DNA methyltransferase inhibitor, a histone demethylase inhibitor or a histone methylation inhibitor. The epigenetic agent may be azacitidine (Vidaza), decitabine (Dacogen), vorinostat (Zoiinza), romidepsin (Istodax) or ruxolitinib (Jakafi). A preferred chemotherapeutic agent that can be combined for prostate cancer treatment is paclitaxel (TAXOL).

[0037] In one embodiment, a kit containing any antigenic therapeutic agent described herein is provided herein.

[0038] When aspects or embodiments of the invention are described with respect to Markush groups or alternative other groupings, the invention encompasses not only all of the recited groups as a whole, but also each member of the group individually and all possible subgroups of the main group and the main group in which one or more of the members of the group are absent. The invention also contemplates any express exclusion of one or more of the group members in the claimed invention. In certain embodiments, for example, the following are provided: (Item 1) An immunogenic vaccine composition comprising a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1 to 6. (Item 2) The immunogenic vaccine composition according to Item 1, wherein the peptide is a synthetic peptide. (Item 3)​ The immunogenic vaccine composition according to item 1, wherein the peptide is a recombinant peptide. (Item 4) The immunogenic vaccine composition according to item 1, wherein the peptide contains a sequence derived from an endogenous retroviral protein. (Item 5) The immunogenic vaccine composition according to item 1, wherein the peptide contains a sequence derived from an exogenous viral protein. (Item 6) The immunogenic vaccine composition according to item 1, wherein the peptide contains a sequence of a protein expressed by cancer cells of a subject having cancer, and the protein is expressed by the cancer cells at a level higher than the level expressed by non-cancer cells of the subject. (Item 7) The immunogenic vaccine composition according to any one of items 1 to 6, wherein the peptide is 100 amino acids in length or less. (Item 8) The immunogenic vaccine composition according to any one of items 1 to 7, wherein the peptide is about 5 to about 50 amino acids in length or about 15 to about 35 amino acids in length. (Item 9) The immunogenic vaccine composition according to any one of items 1 to 8, wherein the peptide is about 30 amino acids in length or less or about 15 amino acids in length or less. (Item 10) The immunogenic vaccine composition according to any one of items 1 to 9, wherein the peptide contains a sequence that binds to major histocompatibility complex (MHC) class I with a binding affinity of less than about 500 nM. (Item 11) The immunogenic vaccine composition according to any one of items 1 to 9, wherein the peptide contains a sequence that binds to major histocompatibility complex (MHC) class II with a binding affinity of less than about 1000 nM. (Item 12) The immunogenic vaccine composition according to any one of items 1 to 11, wherein the peptide further contains a non-natural amino acid adjacent to the at least 8 consecutive amino acids. (Item 13) The immunogenic vaccine composition according to any one of items 1 to 12, wherein the composition further comprises a second peptide comprising at least 8 consecutive amino acids of the sequences in any one of Tables 1 to 6, and the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM. (Item 14) The immunogenic vaccine composition according to item 13, wherein the peptides are linked using a polyglycine or polyserine linker. (Item 15) The immunogenic vaccine composition according to item 13 or 14, wherein the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM or less than about 500 nM. (Item 16) The immunogenic vaccine composition according to any one of items 1 to 15, wherein the peptide further comprises a modification that increases in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. (Item 17) The immunogenic vaccine composition according to item 16, wherein the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation, HESylation, recombinant PEG mimetic, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of an amino acid mimic, or addition of a non-natural amino acid. (Item 18) The immunogenic vaccine composition according to item 16, wherein the peptide comprises a modification that increases targeting by antigen-presenting cells. (Item 19) The immunogenic vaccine composition according to item 18, wherein the antigen-presenting cell is a dendritic cell. (Item 20) The immunogenic vaccine composition according to item 19, wherein the modification that increases the targeting by the dendritic cells is DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, or CD1a marker. (Item 21) The immunogenic vaccine composition according to any one of items 1 to 20, comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 peptides, each containing at least 8 consecutive amino acids of the sequences in any one of Tables 1 to 6. (Item 22) The immunogenic vaccine composition according to any one of items 1 to 20, comprising 2 to 20 peptides, each containing at least 8 consecutive amino acids of the sequences in any one of Tables 1 to 6. (Item 23) The immunogenic vaccine composition according to any one of items 1 to 22, further comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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 or at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 additional antigenic peptides. (Item 24) The immunogenic vaccine composition according to item 23, wherein the additional antigenic peptide is specific to the tumor of an individual patient. (Item 25) The immunogenic vaccine composition according to item 24, wherein the additional antigenic peptide is selected by identifying sequence differences between the genome, exome and / or transcriptome of the patient's tumor sample and the genome, exome and / or transcriptome of a non-tumor sample. (Item 26) The immunogenic vaccine composition according to item 25, wherein identifying the sequence differences comprises performing next-generation sequencing. (Item 27) A composition comprising antigen-presenting cells comprising a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1 to 6. (Item 28) The composition according to item 27, wherein the antigen-presenting cell is a dendritic cell. (Item 29) An in vivo delivery system comprising the composition according to any one of items 1 to 28. (Item 30) The delivery system according to item 29, comprising a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, or liposomes. (Item 31) The delivery system according to item 29, wherein the cell-permeable peptide is a TAT peptide, herpes simplex virus VP22, transportan, or Antp. (Item 32) An immunogenic vaccine composition comprising a recombinant polynucleotide encoding a peptide comprising at least 8 consecutive amino acids of an array in any one of Tables 1 to 6. (Item 33) The immunogenic vaccine composition according to item 32, wherein the recombinant polynucleotide is RNA, and optionally, self-amplifying RNA. (Item 34) The immunogenic vaccine composition according to item 33, wherein the RNA is modified to increase stability, increase cell targeting, increase translation efficiency, adjuvant activity, cytosolic accessibility, and / or decrease cytotoxicity. (Item 35) The immunogenic vaccine composition according to item 34, wherein the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increase in GC content, incorporation of modified nucleosides, incorporation of a 5'-cap or cap analog, and / or incorporation of an unmasked polyA sequence. (Item 36) A composition comprising a cell comprising a recombinant polynucleotide encoding a peptide comprising at least 8 consecutive amino acids of an array in any one of Tables 1 to 6. (Item 37) A composition comprising a vector comprising a polynucleotide comprising a sequence encoding a peptide comprising at least 8 consecutive amino acids of an array in any one of Tables 1 to 6. (Item 38) The composition according to item 37, wherein the polynucleotide is operably linked to a promoter. (Item 39) The composition according to item 37 or 38, wherein the polynucleotide is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion. (Item 40) The composition according to item 39, wherein the virus is an adeno-associated virus, a herpes virus, a lentivirus, or a pseudotype thereof. (Item 41) An in vivo delivery system comprising the composition according to any one of items 32 to 40. (Item 42) The delivery system according to item 41, comprising a spherical nucleic acid, a virus, a virus-like particle, a plasmid, a bacterial plasmid, or a nanoparticle. (Item 43) A T cell receptor (TCR) that specifically binds to a peptide:MHC complex, wherein the peptide of the peptide:MHC complex comprises at least 8 consecutive amino acids of a sequence in any one of Tables 1 to 6. (Item 44) A T cell comprising a T cell receptor (TCR) that specifically binds to a peptide:MHC complex, wherein the peptide of the peptide:MHC complex comprises at least 8 consecutive amino acids of a sequence in any one of Tables 1 to 6. (Item 45) The T cell according to item 44, which is a helper T cell or a cytotoxic T cell. (Item 46) The T cell according to item 44 or 45, which is an autologous patient T cell. (Item 47) A method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject the composition according to any one of items 1 to 28 and 32 to 40; the delivery system according to any one of items 29 to 31, 41 and 42; the TCR according to item 43; or the T cell according to any one of items 44 to 46, wherein the subject comprises cancer cells expressing a protein comprising at least 8 consecutive amino acids of a sequence in any one of Tables 1 to 6. (Item 48) The method according to item 47, wherein the subject is a human. (Item 49) The method according to item 47 or 48, wherein the cancer is selected from the group consisting of urogenital cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, triple-negative breast cancer (TNBC), malignant melanoma, Merkel cell carcinoma or osteosarcoma, hematological neoplasm, multiple myeloma, smoldering myeloma (SMM), acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-insensitive prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, cholangiocarcinoma, head and neck squamous cell cancer, soft tissue sarcoma, and small cell lung cancer. (Item 50) The method according to any one of items 47 to 49, further comprising administering to the subject at least one immune checkpoint inhibitor. (Item 51) The method according to item 50, wherein the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

Mode for Carrying Out the Invention

[0039] Detailed Description This specification describes a novel immunotherapeutic agent and its use based on the discovery of non-mutated protein epitopes expressed in cancer cells. Thus, the present invention described in this specification provides a peptide, a polynucleotide encoding the peptide, and a peptide binder that can be used, for example, to stimulate an immune response against a tumor-associated antigen and to produce an immunogenic composition or a cancer vaccine for use in treating a disease.

[0040] I. Definitions To facilitate understanding of the present invention, some terms and expressions are defined below.

[0041] "Non-mutated protein antigen" refers to an antigen that is specifically expressed, or expressed at a higher level than in non-cancerous tissues, in cancer. These include, but are not limited to, antigens of exogenous viruses, antigens of endogenous retroviruses, and overexpressed antigens without somatic mutations.

[0042] "Viral antigen" refers to an antigen encoded by an exogenous virus.

[0043] "Retroviral antigen" refers to an antigen encoded by an endogenous retroviral sequence.

[0044] "Non-mutated overexpressed antigen" refers to a non-mutated antigen encoded by the genome of cancer cells that is expressed at a higher level than in non-cancerous tissues.

[0045] "Tumor-specific epitope" refers to an epitope that is not expressed in non-cancerous cells or germline cells, but is found to be expressed in cancer cells, or is expressed at a higher level in cancer cells than in non-cancerous cells.

[0046] "Reference" can be used to associate and compare the results obtained in the method of the present invention from tumor specimens. Typically, a "reference" can be obtained based on one or more normal specimens, particularly specimens obtained from a patient or one or more different individuals, such as healthy individuals, particularly individuals of the same species, that are not affected by cancer disease. A "reference" can be determined empirically by testing a sufficient number of normal specimens.

[0047] The term "mutation" refers to a change or difference (nucleotide substitution, addition or deletion) in a nucleic acid sequence as compared to a reference. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and thus do not transfer to offspring. These changes can (but do not always) result in cancer or other diseases. In some embodiments, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a nucleotide substitution that results in an amino acid change, such as an amino acid substitution in a translation product.

[0048] Throughout this disclosure, "binding data" results can be expressed with respect to "IC 50 ". IC 50 is the concentration of the tested peptide at which 50% inhibition of the binding of the labeled reference peptide is observed in a binding assay. Taking into account the conditions under which the assay is performed (i.e., limiting the HLA protein and labeled reference peptide concentrations), these values approximate the K D values. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO94 / 20127 and WO94 / 03205, and in Sidney et al., Current Protocols in Immunology, page 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); and Sette et al., Mol. Immunol. 31:813 (199 4). Alternatively, binding can also be expressed as compared to binding by a reference standard peptide. For example, it can be based on its IC 50 compared to the IC 50 of the reference standard peptide.

[0049] Binding can be determined in live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using surfactant solubilizates (e.g., Cerundolo et al., J. Immunol. 21: 2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assay (Hammer et al., J. Exp. Med. 180:2353 (1994)) and the measurement of class I MHC stabilization or aggregation (e.g., Ljunggren et al., Nature 346:47 6 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol.149:1896 (1992)) can also be determined using other assays.

[0050] "Cross-reactive binding" indicates that a peptide is bound by more than one HLA molecule, and a synonym is degenerate binding.

[0051] When used to consider an epitope, the term "derived from" is synonymous with "prepared from". An epitope derived from may be isolated from a natural source or may be synthesized according to standard protocols in the art. Synthetic epitopes may contain artificial amino acid residues "amino acid mimics" such as D-isomers of naturally occurring L-amino acid residues or non-natural amino acid residues such as cyclohexylalanine. An epitope derived from or prepared from may be an analog of a natural epitope.

[0052] "Diluent" includes water and a sterilizing solution such as an oil of petroleum, animal, plant or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is also a diluent for pharmaceutical compositions. Saline and aqueous glucose and glycerol solutions can also be used, for example, as diluents in injectable solutions.

[0053] "Epitope" is the collective characteristic of molecules such as primary, secondary and tertiary peptide structures and charges that together form a site recognized by, for example, immunoglobulins, T cell receptors, HLA molecules or chimeric antigen receptors. Alternatively, an epitope can be defined as a set of amino acid residues involved in recognition by a specific immunoglobulin, or as residues required for recognition by a T cell receptor protein, chimeric antigen receptor and / or major histocompatibility complex (MHC) receptor in the context of T cells. Epitopes may be prepared by isolation from natural sources or they may be synthesized according to standard protocols in the art. Synthetic epitopes may include D-isomers of naturally occurring L-amino acid residues or artificial amino acid residues such as non-naturally occurring amino acid residues such as cyclohexylalanine, "amino acid mimics". Throughout this disclosure, epitopes may in some cases be referred to as peptides or peptide epitopes.

[0054] It is understood that proteins or peptides comprising an epitope or analog and additional amino acid(s) as described herein are still within the scope of the present invention. In certain embodiments, the peptide comprises a fragment of an antigen.

[0055] In certain embodiments, there are limitations on the peptide length of the present invention. Embodiments with limited length occur when a protein or peptide containing an epitope described herein contains a region (i.e., a continuous series of amino acid residues) that has 100% identity with the native sequence. For example, for the entire native molecule, to avoid defining epitopes from the reading frame, there are limitations on the length of any region having 100% identity with the native peptide sequence. Thus, for peptides containing an epitope described herein and a region having 100% identity with the native peptide sequence, the region having 100% identity with the native sequence generally has a length of 600 amino acid residues or less, 500 amino acid residues or less, 400 amino acid residues or less, 250 amino acid residues or less, 100 amino acid residues or less, 85 amino acid residues or less, 75 amino acid residues or less, 65 amino acid residues or less, and 50 amino acid residues or less. In certain embodiments, the "epitope" described herein is less than 51 amino acid residues having 100% identity with the native peptide sequence in any increment up to 5 amino acid residues, e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue, and is contained by a peptide having such a region.

[0056] "Human leukocyte antigen" or "HLA" are human class I or class II major histocompatibility complex (MHC) proteins (see, e.g., Stites et al., IMMUNOLOGY, 8th ed., Lange Publishing, Los Altos, Calif. (1994)).

[0057] As used herein, "HLA supertype or HLA family" refers to a set of HLA molecules classified based on shared peptide-binding specificities. HLA class I molecules that share a binding affinity similar to a certain degree for peptides carrying a specific amino acid motif are classified into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLAxx-like molecules (where "xx" indicates a specific HLA type) are synonyms.

[0058] The term "identical" or percent "identity", when related to two or more peptide sequences or antigenic fragments, refers to two or more sequences or subsequences that, when compared or aligned for maximum match over a comparison window, using a sequence comparison algorithm or by manual alignment and visual inspection, are the same or have a specified percentage of amino acid residues that are the same.

[0059] An "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" is a peptide that contains an allele-specific motif such that the peptide binds to an HLA molecule and induces a cell-mediated or humoral response, such as a cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and / or B lymphocyte response. Thus, the immunogenic peptides described herein can bind to appropriate HLA molecules, thereby inducing a CTL (cytotoxic) response or HTL (and humoral) response to the peptide.

[0060] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen-binding protein that includes an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, the "constant domain" of a TCR polypeptide includes the membrane-proximal TCR constant domain and may also include the TCR transmembrane domain and / or the TCR cytoplasmic tail. For example, in some embodiments, the CAR is a dimer comprising a first polypeptide that includes an immunoglobulin heavy chain variable domain linked to a TCR beta constant domain, and a second polypeptide that includes an immunoglobulin light chain variable domain (e.g., a kappa or lambda variable domain) linked to a TCR alpha constant domain. In some embodiments, the CAR is a dimer comprising a first polypeptide that includes an immunoglobulin heavy chain variable domain linked to a TCR alpha constant domain, and a second polypeptide that includes an immunoglobulin light chain variable domain (e.g., a kappa or lambda variable domain) linked to a TCR beta constant domain.

[0061] The terms "isolated" or "biologically pure" refer to a substance that substantially or essentially does not contain the components that are normally associated with the substance when found in its natural state. Thus, an isolated peptide described herein does not contain some or all of the substances that are normally associated with the peptide in its native environment. An "isolated" epitope refers to an epitope that does not include the entire sequence of the antigen from which the epitope is derived. Typically, an "isolated" epitope is not bound to additional amino acid residues that result in a sequence having 100% identity over the full length of the native sequence. The native sequence may be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term "isolated" means that the substance has been removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector and / or such a polynucleotide or peptide may be part of a composition, and further, such a vector or composition is "isolated" in that it is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically.

[0062] The "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in the control of cellular interactions that result in a physiological immune response. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see Paul, FUNDAMENTAL IMMUNOLOGY, 3rd Edition (Suppl.), Raven Press, New York (1993).

[0063] A "natural" or "wild-type" sequence refers to a sequence found in nature. Such sequences may include longer sequences in nature.

[0064] "T cell epitope" is understood to mean a peptide sequence that can be bound by class I or class II MHC molecules in the form of peptide-presenting MHC molecules or MHC complexes, and can then be recognized and bound by cytotoxic T lymphocytes or helper T cells respectively in this form.

[0065] "Receptor" is understood to mean a group of molecules that can bind to biological molecules or ligands. Receptors can function to transmit information in cells, cell formations or organisms. Receptors include, for example, at least one receptor unit where each receptor unit can consist of a protein molecule. Receptors have a structure that complements the structure of the ligand and can complex with the ligand as a binding partner. Information is transmitted particularly by conformational changes of the receptor following complex formation of the ligand at the cell surface. In some embodiments, the receptor is particularly understood to mean a ligand, and in particular a peptide or peptide fragment of a suitable length, and proteins of MHC class I and II that can form a receptor / ligand complex.

[0066] "Ligand" is understood to mean a molecule having a structure that is complementary to the structure of a receptor and can complex with this receptor. In some embodiments, the ligand is understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence, whereby the peptide or peptide fragment can complex with a protein of MHC class I or MHC class II.

[0067] In some embodiments, "receptor / ligand complex" is also understood to mean "receptor / peptide complex" or "receptor / peptide fragment complex" containing a peptide or peptide fragment presenting a class I or class II MHC molecule.

[0068] The terms "major histocompatibility complex (MHC) protein or molecule", "MHC molecule", "MHC protein" or "HLA protein" are understood to mean proteins that can bind to peptides resulting from the proteolytic cleavage of protein antigens, can present promising lymphocyte epitopes (such as T cell epitopes and B cell epitopes), can transport them to the cell surface, and can present them to specific cells, particularly cytotoxic T lymphocytes, helper T cells or B cells. The major histocompatibility complex in the genome contains genetic regions whose gene products expressed on the cell surface are important for the binding and presentation of endogenous and / or foreign antigens and thereby for regulating immunological processes. The major histocompatibility complex is classified into two gene groups encoding different proteins, namely molecules of MHC class I and molecules of MHC class II. The cell biology and expression patterns of the two MHC classes are adapted to these various roles.

[0069] The terms "peptide" and "peptide epitope" are used interchangeably herein as "oligopeptide" to denote a series of residues connected to each other by peptide bonds typically between the α-amino and carboxyl groups of adjacent amino acid residues.

[0070] "Synthetic peptide" refers to a peptide obtained from a non-natural source, for example one that is artificial. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion proteins".

[0071] "PanDR-binding" peptides, "PanDR-binding epitopes" are members of a family of molecules that bind to more than one HLA class II DR molecule.

[0072] "Pharmaceutically acceptable" refers generally to compositions or components of compositions that are non-toxic, inert and / or physiologically compatible.

[0073] "Pharmaceutical excipient" or "excipient" includes substances such as adjuvants, carriers, pH regulators and buffers, osmotic pressure regulators, wetting agents, preservatives, etc. "Pharmaceutical excipient" is a pharmaceutically acceptable excipient.

[0074] The term "motif" refers to a pattern of residues of a defined length in an amino acid sequence, for example, for class I HLA motifs recognized by a particular HLA molecule, less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, about 8 to about 13 (e.g., 8, 9, 10, 11, 12 or 13) amino acid residues, and for class II HLA motifs about 6 to about 25 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acid residues of a peptide. Motifs typically differ for each HLA protein encoded by a given human HLA allele. These motifs differ in the pattern of primary and secondary anchor residues. In some embodiments, the MHC class I motif identifies peptides that are 9, 10 or 11 amino acid residues in length.

[0075] "Supermotif" is a peptide-binding specificity shared by HLA molecules encoded by two or more HLA alleles. In some embodiments, the supermotif-bearing peptides described herein are recognized with high or moderate affinity (as defined herein) by two or more HLA antigens.

[0076] As used herein, the term "naturally occurring" refers to the fact that the object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by a person in a laboratory is naturally occurring.

[0077] According to the present invention, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or product that, upon administration, induces an immune response, such as a cellular or humoral immune response that recognizes and attacks disease cells such as pathogens or cancer cells. Vaccines can be used for the prevention or treatment of diseases. The term "cancer vaccine tailored to an individual" or " individualized cancer vaccine" relates to a specific cancer patient and means that the cancer vaccine is adapted to the requirements or special circumstances of the individual cancer patient.

[0078] "Defensive immune response" or "therapeutic immune response" refers to CTL and / or HTL responses to antigens derived from pathogenic antigens (e.g., tumor antigens) that prevent or at least partially inhibit disease symptoms, side effects, or progression in some way. The immune response may also include an antibody response promoted by the stimulation of helper T cells.

[0079] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into processing products that are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides), and the association (e.g., via binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells, such as antigen-presenting cells.

[0080] An "antigen-presenting cell" (APC) is a cell that presents peptide fragments of protein antigens associated with MHC molecules on their cell surfaces. Some APCs activate antigen-specific T cells. Professional antigen-presenting cells are very efficient at internalizing antigens by phagocytosis or receptor-mediated endocytosis and then displaying fragments of the antigen bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Subsequently, additional co-stimulatory signals are produced by the antigen-presenting cell, resulting in the activation of the T cell. The expression of co-stimulatory molecules characterizes professional antigen-presenting cells.

[0081] The main types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which have the broadest antigen presentation and are probably the most important antigen-presenting cells.

[0082] Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both the MHC class II and I antigen presentation pathways. It is well known that dendritic cells are powerful inducers of the immune response, and activation of these cells is an essential step for the induction of antitumor immunity.

[0083] Dendritic cells can be conveniently categorized as "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding possible intermediate stages of differentiation.

[0084] Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, associated with high expression of Fc receptors and mannose receptors. The mature phenotype is typically characterized by low expression of these markers but high expression of cell surface molecules that cause T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0085] The term "residue" refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or to a nucleic acid (DNA or RNA) encoding an amino acid or amino acid mimetic.

[0086] The nomenclature used to describe peptides or proteins follows the conventional convention in which the amino group (amino or N-terminus) is represented on the left side of each amino acid residue and the carboxyl group (carboxy or C-terminus) is represented on the right side. When the positions of amino acid residues are referred to in a peptide epitope, they are numbered from amino to carboxyl direction, and the residue located at the amino terminus of the epitope or peptide or the protein, which it may be a part of, is position 1.

[0087] In the formulas representing selected specific embodiments of the present invention, the amino-terminal group and the carboxyl-terminal group are in the form estimated at physiological pH values, even if not specifically shown, unless otherwise specified. In the amino acid structural formula, each residue is generally represented by the standard three-letter or one-letter notation. The L-form of an amino acid residue is represented by a single capital letter or the first capital letter of the three-letter symbol, and the D-form of an amino acid residue having the D-form is represented by a single lowercase letter or a three-letter symbol in lowercase. However, when three-letter symbols or full names are used without including capital letters, they refer to L-amino acid residues. Glycine has no asymmetric carbon atom and is simply shown as "Gly" or "G". The amino acid sequences of the peptides described herein are generally written using the standard one-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0088] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments, the polynucleotide administered is mRNA.

[0089] The term "identical" or percent "identity" in reference to two or more nucleic acids or polypeptides refers to two or more sequences or subsequences having the specified percentage of nucleotide or amino acid residues that are the same or that would be the same when compared for maximum correspondence and aligned (introducing gaps if necessary) without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining an alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical if, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40 - 60 residues, at least about 60 - 80 residues or any integer between the two values in length. In some embodiments, identity exists over a region longer than 60 - 80 residues, such as at least about 80 - 100 residues, and in some embodiments the sequences are substantially identical over the entire length of the sequences being compared, such as the coding region of a nucleotide sequence.

[0090] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate peptide function are well known in the art.

[0091] As used herein, the term "vector" means a construct capable of delivering and usually expressing one or more genes and / or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents and DNA or RNA expression vectors encapsulated in liposomes.

[0092] An "isolated" polypeptide, antibody, polynucleotide, vector, cell or composition is a polypeptide, antibody, polynucleotide, vector, cell or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that are purified to a degree that they are no longer in the form found in nature. In some embodiments, the isolated polypeptide, antibody, polynucleotide, vector cell or composition is substantially pure. In one embodiment, an "isolated polynucleotide" includes a PCR or quantitative PCR reaction product that includes a polynucleotide amplified in a PCR or quantitative PCR reaction.

[0093] As used herein, the term "substantially pure" refers to a substance that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure.

[0094] The term "subject" refers to any animal (e.g., a mammal) including, but not limited to, a human, non-human primate, dog, cat, rodent, etc. that is the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0095] The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to an amount that is therapeutically effective to "treat" a disease or disorder in a subject or mammal. The therapeutically effective amount of a drug has a therapeutic effect and can itself prevent the onset of a disease or disorder, delay the onset of a disease or disorder, slow the progression of a disease or disorder, alleviate to some extent one or more symptoms associated with the disease or disorder, reduce morbidity and mortality, improve quality of life, or can be a combination of such effects.

[0096] The terms "treating", "treatment", "to treat", "alleviating", or "to alleviate" refer to both 1) therapeutic means of curing, retarding, alleviating, and / or arresting the progression of the symptoms of a diagnosed pathological condition or disorder, and 2) prophylactic or preventative means of preventing or delaying the onset of a targeted pathological condition or disorder. Accordingly, those in need of treatment include those already having a disorder, those prone to having a disorder, and those in which the disorder is being prevented.

[0097] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0098] For example, it is understood that terms such as "comprises", "comprised", "comprising" may have the meaning ascribed to them under United States patent law, e.g., they can mean "includes", "included", "including", etc., and terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them under United States patent law, e.g., they permit elements not expressly recited but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention. The description herein is not intended to be limiting.

[0099] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in expressions such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0100] II. Non-mutated protein antigens expressed in cancer cells The applicants have discovered antigens expressed by cancer cells encoded by the following genes: ERVH-2 matrix protein, ERVH-2 gag, ERVH48-1 coat protein, ERVH48-1 syncytin, ERVE-4 reverse transcriptase, ERVK-5 gag, env, pol proteins, and ERVI-1 envelope protein.

[0101] The applicants have discovered antigens expressed by cancer encoded by the following genes: TYR, MAGEC1, MAGEA10, MAGEB17, MAGEA4, MABEB16, MAGEA1, MAGEA8, MAGEB4, CT45A5, ALPPL2, MMP13, CTAG1B, DCT, CLDN6, MLANA, AFP, DKK4, ASCL2, GAGE1, GAGE10, SLC45A2, PAGE5, PAGE2, and PMEL.

[0102] The applicants have discovered antigens expressed by cancer encoded by the following genes: HPV-16, E6, HPV-16 E7, EBV LF2, EBV BALF5, EBV RPMS1, EBV A73, EBV BALF4, EBV BALF3, and EBV BARF0. Non-mutated protein epitope polypeptides

[0103] In one aspect, the present invention provides an isolated peptide comprising a non-mutated protein epitope expressed in cancer cells. In some embodiments, the non-mutated protein epitope is a retroviral antigen. In some embodiments, the non-mutated protein epitope is a non-mutated overexpressed antigen. In some embodiments, the non-mutated protein epitope is a viral antigen.

[0104] In one aspect, the present invention provides an isolated peptide comprising a peptide derived from Tables 1-6. The term "peptide" is typically used herein to designate a series of residues, typically L-amino acids, connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, the term "polypeptide" is typically used herein to designate a series of residues, such as L-amino acids, connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The polypeptide or peptide may be of various lengths and may be in its neutral (uncharged) form or in the form of a salt, and may or may not contain modifications such as glycosylation, side chain oxidation, or phosphorylation, or may be subject to conditions under which these modifications do not destroy the biological activity of the polypeptides described herein.

[0105] In some embodiments, sequencing methods are used to identify tumor-specific epitopes. Any suitable sequencing method, such as next-generation sequencing (NGS) technology, can be used according to the present invention. Third-generation sequencing methods may replace NGS technology in the future to accelerate the sequencing step of the method. For clarity, the term "next-generation sequencing" or "NGS" in the context of the present invention means all new high-efficiency sequencing technologies that randomly read nucleic acid templates in parallel along the entire genome by breaking the entire genome into small pieces, as opposed to the "conventional" sequencing method known as Sanger chemistry. Such NGS technologies (also known as massively parallel sequencing technologies) can deliver nucleic acid sequence information of the entire genome, exome, transcriptome (all transcribed sequences of the genome), or methylome (all methylated sequences of the genome) in a very short period of time, for example, within 1-2 weeks, for example, within 1-7 days or within less than 24 hours, and in principle enable single-cell sequencing methods. A plurality of NGS platforms that are commercially available or described in the literature, such as those detailed in WO2012 / 159643, can be used in the context of the present invention.

[0106] In certain embodiments, the non-mutated protein epitope peptides described herein may include, but are not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therefrom. In certain embodiments, the non-mutated protein epitope peptide molecule is equal to or less than 100 amino acids.

[0107] In some embodiments, the non-mutated protein epitope peptides and polypeptides described herein with respect to MHC class I are 13 residues or less in length, typically between about 8 and about 11 residues, and in particular, consist of 9 or 10 residues. In some embodiments, the non-mutated protein epitope peptides and polypeptides described herein with respect to MHC class II are 9 to 24 residues in length.

[0108] Longer non-mutated protein epitope peptides can be designed in several ways. In some embodiments, when the HLA-binding peptides are predicted or known, the longer non-mutated protein epitope peptides can consist of (1) individual binding peptides having extensions of 2 - 5 amino acids towards the N-terminus and C-terminus of each corresponding peptide; or (2) a partial or complete ligation of binding peptides containing extended sequences for each. In some embodiments, the use of longer peptides is presumed to allow for endogenous processing by the patient's cells and can result in more efficient antigen presentation and induction of T cell responses. In some embodiments, when peptides overlap and are displayed side by side on a long non-mutated protein epitope peptide, two or more peptides can be used.

[0109] In some embodiments, the non-mutated protein epitope peptides and polypeptides bind to an HLA protein (e.g., HLA class I or HLA class II). In certain embodiments, the non-mutated protein epitope peptide or polypeptide has an IC 50 less than at least 5000 nM, less than at least 500 nM, less than at least 100 nM, less than at least 50 nM or less.

[0110] In some embodiments, the non-mutated protein epitope peptides described herein may include carriers such as carriers well-known in the art, for example, albumin such as thyroglobulin, human serum albumin, tetanus toxoid, polyamino acid residues such as poly-L-lysine, poly-L-glutamic acid, influenza virus protein, hepatitis B virus core protein, and the like.

[0111] In some embodiments, the non-mutated protein epitope peptides described herein are terminal -NH 2 acylated, for example, by alkanoyl (C 1 ~C 20 ) or thioglycolyl acetylation, terminal carboxyl amidation, for example, by ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linking to a support or other molecules.

[0112] In some embodiments, the non-mutated protein epitope peptides described herein may contain modifications such as, but not limited to, glycosylation, side-chain oxidation, biotinylation, phosphorylation, addition of surfactants such as lipids, or can be chemically modified, for example, by acetylation. Further, the bonds in the peptide may be other than peptide bonds, for example, covalent bonds, ester bonds or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0113] In some embodiments, the non-mutated protein epitope peptides described herein may contain substitutions to modify the physical properties (e.g., stability or solubility) of the resulting peptides. For example, the non-mutated protein epitope peptides can be modified by substitution of cysteine (C) with α-aminobutyric acid (“B”). Due to its chemical nature, cysteine has a tendency to form disulfide bridges, structurally altering the peptide sufficiently to reduce binding ability. Substitution of α-aminobutyric acid for C not only alleviates this problem but, in certain instances, actually improves binding and cross-linking capabilities. Substitution of cysteine with α-aminobutyric acid can occur at any residue of the non-mutated protein epitope peptide, for example, at the anchor or non-anchor positions of the epitope or analog within the peptide, or at other positions of the peptide.

[0114] In some embodiments, the non-mutated protein epitope peptides described herein are amino acid mimetics or non-natural amino acid residues such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1-, -2-, -3-, or 4-pyrenylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L- (3-Pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine may be included, where the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isobutyl, iso-pentyl, or a non-acidic amino acid residue. Examples of the aromatic ring of the unnatural amino acid include thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides having various amino acid mimics or unnatural amino acid residues are particularly useful because they tend to exhibit increased stability in vivo. Such peptides also have improved storage periods or manufacturing characteristics.

[0115] Peptide stability can be assayed in several ways. For example, various biological media such as peptidases and human plasma and serum have been used to test stability. For example, Verhoef et al., Eur. J. Drug Metab.Pharmacokinetics Vol. 11 : See page 291 (1986). The half-life of the peptides described herein is conveniently determined using a 25% human serum (v / v) assay. The protocol is as follows: Pooled human serum (AB type, non-heat inactivated) is disrupted by centrifugation prior to use. The serum is then diluted to 25% using RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of the reaction solution is removed and added to 6% aqueous trichloroacetic acid (TCA) or ethanol. The turbid reaction sample is cooled (4 °C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatography conditions.

[0116] In some embodiments, the non-mutated protein epitope peptides described herein may be in solution, lyophilized, or in crystalline form.

[0117] In some embodiments, the non-mutated protein epitope peptides described herein can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or isolated from natural sources such as natural tumors or pathogenic organisms. The epitopes can be synthesized individually or conjugated directly or indirectly in the peptide. The non-mutated protein epitope peptides described herein are substantially free of other naturally occurring host cell proteins and fragments thereof, although in some embodiments the peptides can be synthetically conjugated to be conjugated to natural fragments or particles.

[0118] In some embodiments, the non-mutated protein epitope peptides described herein can be prepared in a variety of ways. In some embodiments, the peptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols (see, e.g., Stewart and Young, SOLID PHASE PEPTIDE SYNTHESIS, 2nd ed., Pierce Chemical Co., 1984). Further, individual peptides can be joined using chemical ligation to produce larger peptides that are still within the scope of the invention.

[0119] Alternatively, recombinant DNA techniques can be used in which a nucleotide sequence encoding the peptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression. These procedures are generally known in the art as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant peptides containing or consisting of one or more epitopes described herein can be used to present suitable T cell epitopes.

[0120] In one aspect, the invention described herein also provides a composition comprising one, at least two, or more than two non-mutated protein epitope peptides. In some embodiments, the compositions described herein contain at least two different peptides. In some embodiments, at least two different peptides are derived from the same polypeptide. Different polypeptides means that the peptides differ in length, amino acid sequence, or both. The peptides are derived from any polypeptide known or found to contain tumor-specific epitopes.

[0121] Non-mutated protein epitope polynucleotide Polynucleotides encoding each of the peptides described herein are also part of the present invention. As will be understood by those skilled in the art, due to the redundancy of the genetic code, various nucleic acids encode the same peptide. Each of these nucleic acids is within the scope of the present invention. This embodiment of the present invention includes DNA and RNA, such as mRNA, and in certain embodiments, combinations of DNA and RNA. In one embodiment, the mRNA is self-amplifying mRNA (Brito et al., Adv. Genet. 2015; 89: 179-233). It should be understood that any polynucleotide encoding the peptides described herein is within the scope of the present invention. It should be understood that any polynucleotide encoding the peptides described herein is within the scope of the present invention.

[0122] The term "RNA" includes "mRNA" and, in some embodiments, relates to "mRNA". The term "mRNA" means "messenger RNA" and relates to a "transcript" generated by using a DNA template and encoding a peptide or polypeptide. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In one embodiment, the mRNA is self-amplifying mRNA. In the context of the present invention, mRNA can be generated by in vitro transcription from a DNA template. Methods of in vitro transcription are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0123] The stability and translation efficiency of the RNA can be modified according to requirements. For example, the RNA can be stabilized and its translation increased by one or more modifications that have an RNA stabilizing effect and / or increase the translation efficiency of the RNA. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of the RNA used according to the present invention, it is modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without changing the sequence of the expressed peptide or protein, to increase the GC content, increase the stability of the mRNA, perform codon optimization, and thus enhance translation in the cell.

[0124] The term "modification" in the context of the RNA used in the present invention includes any modification of the RNA that is not naturally present in said RNA. In one embodiment of the present invention, the RNA used according to the present invention does not have an uncapped 5'-triphosphate. Removal of such an uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase. The RNA according to the present invention may have modified ribonucleotides to increase its stability and / or decrease its cytotoxicity. For example, in one embodiment, 5-methylcytidine in the RNA used according to the present invention is partially or completely, for example, completely, replaced with cytidine. Alternatively, or in addition, in one embodiment, pseudouridine in the RNA used according to the present invention is partially or completely, for example, completely, replaced with uridine.

[0125] In one embodiment, the term "modified" relates to providing an RNA having a 5' cap or 5' cap analog. The term "5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide connected to the mRNA by an unusual 5'-5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7 position. The term "conventional 5' cap" refers to the naturally occurring RNA 5' cap, the 7-methylguanosine cap (mG). In the context of the present invention, the term "5' cap" includes 5' cap analogs that are modified to be similar to the RNA cap structure and have the ability to stabilize RNA and / or enhance translation of the RNA when bound to it in vivo and / or in cells.

[0126] In certain embodiments, an mRNA encoding a non-mutated protein epitope is administered to a subject in need thereof. In one embodiment, the present invention provides RNAs, oligoribonucleotides, and polynucleotide molecules containing modified nucleosides, gene therapy vectors containing the same, gene therapy methods, and gene transcription silencing methods containing the same. In one embodiment, the administered mRNA contains at least one modified nucleoside.

[0127] The polynucleotides encoding the peptides described herein can be synthesized by chemical techniques, such as the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides containing or consisting of analogs can be readily prepared by substituting appropriate and desired nucleobase(s) in place of those encoding the native epitope.

[0128] ​A number of vectors and host systems suitable for generating and administering the non-mutated protein epitope peptides described herein are known to those skilled in the art and are commercially available. By way of example, the following vectors are provided. Bacteria: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pCR (Invitrogen). Eukaryotes: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

[0129] Representative examples of suitable hosts include bacterial cells such as E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus; fungal cells such as yeast; insect cells such as Drosophila and Sf9; Gluzman , COS-7 line of monkey kidney fibroblasts described by Cell 23: 175 (1981), and other cell lines capable of expressing equivalent vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines or animal cells such as Bowes melanoma; plant cells and the like can be mentioned. The selection of a suitable host is considered to be within the scope of those skilled in the art from the teachings herein.

[0130] Accordingly, the present disclosure also relates to vectors useful for the production and administration of the non-mutated protein epitope peptides described herein, and expression vectors, and host cells containing such vectors.

[0131] The host cell is genetically engineered (transduced, transformed, or transfected) using a vector which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a virus particle, a phage, etc. The engineered host cell can be cultured in a conventional nutrient medium appropriately modified for activating the promoter, selecting transformants, or amplifying the polynucleotide. For example, the culture conditions such as temperature, pH, etc. are those previously used for the host cell selected for expression and will be apparent to those skilled in the art.

[0132] For the expression of the non-mutated protein epitope peptides described herein, start and stop codons, promoter and terminator regions, and in some embodiments, a coding sequence operably linked to a replication system for providing an expression vector for expression in a desired cell host are provided. For example, a promoter sequence compatible with a bacterial host is provided in a plasmid containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vector is transformed into a suitable bacterial host.

[0133] Generally, a recombinant expression vector contains an origin of replication and a selectable marker enabling transformation of a host cell, such as the ampicillin resistance gene of E. coli and the TRP1 gene of S. cerevisiae, as well as a promoter derived from a highly expressed gene for directing transcription of downstream structural sequences. Such promoters can be derived, inter alia, from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock protein. The heterologous structural sequences are assembled at an appropriate stage together with translation initiation and termination sequences, and in some embodiments, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence may encode a fusion protein containing an N-terminal identification peptide conferring desired features, such as stabilization of the expressed recombinant product or simplified purification.

[0134] Also, suitable vectors and control sequences can be used to employ yeast, insect, or mammalian cell hosts. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts described by Gluzman, Cell 23:175 p. (1981), and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines. Mammalian expression vectors include an origin of replication, suitable promoters and enhancers, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences. Such promoters can also be of viral origin, for example, from the human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type 1 (HSV TK promoter). Nucleic acid sequences derived from SV40 splicing and polyadenylation sites can be used to provide non-transcribed gene elements.

[0135] The polynucleotides encoding the non-mutated protein epitope peptides described herein may also contain ubiquitination signal sequences that facilitate the movement of the resulting peptides into the endoplasmic reticulum, and / or targeting sequences such as endoplasmic reticulum (ER) signal sequences.

[0136] The polynucleotides described herein can be administered to and expressed in human cells (e.g., immune cells including dendritic cells). The human codon usage table can be used to guide codon selection for each amino acid. Such polynucleotides may contain spacer amino acid residues between epitopes and / or analogs, such as those described above, or may contain naturally occurring flanking sequences adjacent to the epitopes and / or analogs (and / or CTL, HTL, and B cell epitopes).

[0137] In some embodiments, the non-mutated protein epitope peptides described herein can also be administered / expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector for expressing the nucleotide sequence encoding the non-mutated protein epitope peptide described herein. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described by Stover et al., Nature It is described by Volume 351: pages 456 - 460 (1991). Various other vectors useful for the therapeutic administration or immunization of the non - mutated protein epitope polypeptides described herein, such as adenovirus and adeno - associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, Sendai virus vectors, poxvirus vectors, canarypox vectors, and fowlpox vectors, etc., will be apparent to those skilled in the art from the description herein. In some embodiments, the vector is a modified vaccinia Ankara (VA) (e.g., Bavarian Noridic (MVA - BN)).

[0138] Standard regulatory sequences well - known to those skilled in the art can be included in the vector to ensure expression in human target cells. Several vector elements are desirable: a polynucleotide, e.g., a promoter having a downstream cloning site for mini - gene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Several promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. For other suitable promoter sequences, see, for example, U.S. Patents Nos. 5,580,859 and 5,589,466. In some embodiments, the promoter is the CMV - IE promoter.

[0139] The polynucleotides described herein may contain one or more synthetic or naturally - occurring introns in the transcribed region. The inclusion of mRNA - stabilizing sequences and sequences for replication in mammalian cells can also be considered to increase polynucleotide expression.

[0140] Furthermore, the polynucleotides described herein may contain immunostimulatory sequences (ISS or CpG). These sequences can be included outside of the polynucleotide coding sequence in the vector to enhance immunogenicity.

[0141] Non-mutated protein epitope-binding peptide In certain embodiments, the invention provides a binding protein (e.g., an antibody or an antigen-binding fragment thereof), or a T cell receptor (TCR), or a chimeric antigen receptor (CAR) that can bind with high affinity to a non-mutated protein epitope peptide: human leukocyte antigen (HLA) complex. In some embodiments, the invention provides a CAR that can bind with high affinity to a non-mutated protein epitope peptide derived from the extracellular domain of a protein. In certain embodiments, the antigen-specific binding protein or TCR or CAR described herein includes variant polypeptide species having one or more amino acid substitutions, insertions, or deletions in the native amino acid sequence, provided that the binding protein retains or substantially retains its specific binding function. Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when recombinantly producing a binding protein or TCR. Amino acid substitutions, deletions, and additions can be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., 2001). Oligonucleotide-directed site-specific Mutagenesis procedures can be used to provide a changed polynucleotide having a specific codon changed according to the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis, can be used to prepare immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0142] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be included in the following categories: amino acids having basic side chains (e.g., lysine, arginine, histidine); amino acids having acidic side chains (e.g., aspartic acid, glutamic acid); amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In certain circumstances, a substitution of glutamine for glutamic acid or asparagine for aspartic acid can be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence of the polypeptide and its conservative amino acid substitutions to the sequence of a second polypeptide (e.g., using the GENEWORKS, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0143] In certain embodiments, a non-mutated protein epitope-specific binding protein, TCR or CAR can specifically bind to an antigen:HLA complex on the cell surface without depending on CD8 or in the absence of CD8. In certain embodiments, the non-mutated protein epitope-specific binding protein is a T cell receptor (TCR), a chimeric antigen receptor or an antigen-binding fragment of a TCR, all of which may be chimeric, humanized or human. In a further embodiment, the antigen-binding fragment of the TCR comprises a single-chain TCR (scTCR).

[0144] In certain embodiments, there is provided a composition comprising a non-mutated protein epitope-specific binding protein or a high-affinity recombinant TCR according to any one of the above embodiments and a pharmaceutically acceptable carrier, diluent, or excipient.

[0145] Methods useful for isolating and purifying recombinantly produced soluble TCRs may include, by way of example, obtaining a supernatant from a suitable host cell / vector system that secretes the recombinant soluble TCR into the culture medium and then concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices such as an affinity matrix or an ion exchange resin. The recombinant polypeptide can be further purified using one or more reverse-phase HPLC steps. These purification methods can also be used when isolating an immunogen from its natural environment. Methods for large-scale production of one or more of the isolated / recombinant soluble TCRs described herein include batch cell culture that is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR can be carried out according to methods described herein and known in the art.

[0146] III. Immunogenic Compositions and Vaccine Compositions In one embodiment, provided herein is an immunogenic composition, e.g., a vaccine composition capable of generating a non-mutated protein epitope-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a non-mutated protein epitope therapeutic agent (e.g., a peptide, polynucleotide, TCR, CAR, a cell containing a TCR or CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.) as described herein that corresponds to a tumor-specific non-mutated protein epitope identified herein.

[0147] One of ordinary skill in the art can analyze, for example, T cell generation in vitro and their efficiency and overall presence, the proliferation, affinity, and expansion of certain T cells for a particular peptide, and the functionality of T cells by, for example, IFN-γ production or tumor killing by T cells to select a non-mutated protein epitope therapeutic agent. The most efficient peptides can then be combined as an immunogenic composition.

[0148] In one embodiment of the invention, various non-mutated protein epitope peptides and / or polypeptides are selected such that one immunogenic composition comprises non-mutated protein epitope peptides and / or polypeptides that can associate with various MHC molecules, e.g., various MHC class I molecules. In some embodiments, the immunogenic composition comprises non-mutated protein epitope peptides and / or polypeptides that can associate with the most frequently occurring MHC class I molecules. Thus, the immunogenic compositions described herein comprise various peptides that can associate with at least two, at least three, or at least four MHC class I or class II molecules.

[0149] In one embodiment, the immunogenic compositions described herein can generate a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0150] In some embodiments, the immunogenic compositions described herein may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein below. The polypeptides and / or polynucleotides in the composition may be associated with a carrier, such as a protein or an antigen-presenting cell, such as a dendritic cell (DC) that can present a peptide to T cells or B cells. In a further embodiment, the DC-binding peptide is used as a carrier for targeting non-mutated protein epitope peptides and polynucleotides encoding non-mutated protein epitope peptides to dendritic cells (Sioud et al. , FASEB J 27:3272-3283 (2013)).

[0151] In embodiments, the non-mutated protein epitope polypeptide or polynucleotide can be provided as an antigen-presenting cell (such as a dendritic cell) containing such a polypeptide or polynucleotide. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient.

[0152] In some embodiments, the antigen-presenting cell is a dendritic cell. In related embodiments, the dendritic cell is an autologous dendritic cell pulsed with a non-mutated protein epitope peptide or nucleic acid. The non-mutated protein epitope peptide can be any suitable peptide that elicits an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides from tumor-associated antigens has been disclosed by Murphy et al. (1996) The Prostate 29 Vol., 371-380 and Tjua et al. (1997) The Prostate 32, 272-2 78. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is an HTL.

[0153] Accordingly, one embodiment of the present invention is an immunogenic composition containing one or more non-mutated protein epitope polypeptides or polynucleotides described in this specification, and pulsed or at least one antigen-presenting cell (e.g., dendritic cell) loaded therewith. In an embodiment, such an APC is autologous (e.g., autologous dendritic cell). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded ex vivo with a non-mutated protein epitope peptide or polynucleotide. In related embodiments, such APCs or PBMCs are injected back into the patient.

[0154] The polynucleotide can be any suitable polynucleotide that can be transduced into dendritic cells and thus result in the presentation of non-mutated protein epitope peptides and the induction of immunity. In one embodiment, the polynucleotide may be naked DNA that is taken up by cells by passive loading. In another embodiment, the polynucleotide is part of a delivery vehicle, such as a liposome, virus-like particle, plasmid, or expression vector. In another embodiment, the polynucleotide is delivered by a vector-free delivery system, such as high-performance electroporation and high-speed cell deformation. In an embodiment, such antigen-presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells). In related embodiments, the T cells are CTLs. In other related embodiments, the T cells are HTLs. Such T cells are then injected into the patient. In some embodiments, CTLs are injected into the patient. In some embodiments, HTLs are injected into the patient. In some embodiments, both CTLs and HTLs are injected into the patient. Administration of any of the therapeutic agents can be done simultaneously, or sequentially and in any order.

[0155] The pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment are intended for parenteral, topical, intranasal, oral, or local administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In embodiments, the composition can be administered intratumorally. The composition may be administered to a surgical resection site to induce a local immune response to the tumor. In some embodiments, compositions for parenteral administration comprising a solution of non-mutated protein epitope peptides are described herein, and the immunogenic compositions are dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. can be used. These compositions may be sterilized by conventional well-known sterilization techniques or may be filter sterilized. The resulting aqueous solution may be packaged as such for use or may be lyophilized, and the lyophilized preparation is combined with a sterile solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH regulators and buffers, osmotic pressure regulators, wetting agents, etc., e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc., as required to approximate physiological conditions.

[0156] The concentration of the non-mutated protein epitope peptides and polynucleotides described herein in the pharmaceutical formulation can vary widely, i.e., from less than about 0.1% by weight to usually or at least about 2% by weight, and up to 20% to 50% by weight or more, and is selected according to the particular mode of administration chosen, depending on fluid volume, viscosity, etc.

[0157] In addition, the non-mutated protein epitope peptides and polynucleotides described herein can be administered via liposomes that target the peptide to specific cell tissues, such as lymphoid tissues. Liposomes are also useful in increasing the half-life of the peptide. Examples of liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the peptide to be delivered can be incorporated as part of the liposome alone, or together with, for example, a molecule that binds to a receptor that is dominant among lymphoid cells, such as a monoclonal antibody that binds to the DEC205 antigen, or together with other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired peptides or polynucleotides described herein can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic / immunogenic polypeptide / polynucleotide composition. Liposomes can generally be formed from standard vesicle-forming lipids, including neutral and charged phospholipids and sterols, such as cholesterol. The choice of lipids is generally guided by considerations such as liposome size, acid lability, and stability of the liposome in the bloodstream. For example, various methods for preparing liposomes can be used as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0158] For targeting immune cells, a non-mutated protein epitope polypeptide or polynucleotide for a cell surface determinant of a desired immune system cell is incorporated into the liposome. The liposome suspension containing the peptide can be administered intravenously, topically, locally, etc. at a dose that varies particularly according to the mode of administration, the polypeptide or polynucleotide to be delivered, and the stage of the disease being treated.

[0159] In some embodiments, the non-mutated protein epitope polypeptide and polynucleotide are targeted to dendritic cells. In one embodiment, the non-mutated protein epitope polypeptide and polynucleotide are targeted to dendritic cells using a marker such as DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, the TSLP receptor or CD1a.

[0160] For solid compositions, conventional or nanoparticle non-toxic solid carriers can be used, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, pharmaceutically acceptable non-toxic compositions are formed by incorporating commonly used excipients, such as any of the carriers listed above, and generally 10 - 95% of the active ingredient, i.e., one or more of the non-mutated protein epitope polypeptides or polynucleotides described herein at a concentration of 25% - 75%.

[0161] For aerosol administration, the non-mutated protein epitope polypeptide or polynucleotide can be supplied in a micronized form with a surfactant and a propellant. Representatives of such agents are esters or partial esters of fatty acids containing 6 - 22 carbon atoms, with an aliphatic polyhydric alcohol or its cyclic anhydride, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid. Mixed esters, such as mixed or natural glycerides may be used. The surfactant may constitute 0.1 wt% - 20 wt% or 0.25 - 5 wt% of the composition. The remainder of the composition may be a propellant. Also, a carrier may be included if desired, such as lecithin for nasal delivery.

[0162] In addition, additional methods for delivering the non-mutated protein epitope polynucleotides described herein are known in the art. For example, nucleic acids can be delivered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Pat. Nos. 5,580,859 and 5,589,466. Also, nucleic acids can be administered using ballistic delivery, as described in, for example, U.S. Pat. No. 5,204,253 administered. Particles consisting of DNA alone may be administered. Alternatively, the DNA may be attached to particles, such as gold particles.

[0163] In addition, for therapeutic or immunological purposes, mRNA encoding a non-mutated protein epitope peptide or peptide binder may be administered to a patient. In one embodiment, the mRNA is self-amplifying RNA. In a further embodiment, the self-amplifying RNA is part of a synthetic lipid nanoparticle formulation (Geall et al., Proc Natl Acad Sci U S A. 109:14604-14609 (2012)).

[0164] Also, nucleic acids can be delivered complexed with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO96 / 18372, WO93 / 24640; Mannino and Gould-Fogerite, BioTechniques 6(7):682-691 (1988); U.S. Pat. No. 5,279,833; WO91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987 year).

[0165] In addition, the non-mutated protein epitope peptides and polypeptides described herein can be expressed by attenuated viruses such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector for expressing the nucleotide sequence encoding the peptides described herein. Upon introduction into an acutely or chronically infected host or a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, thereby inducing a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the therapeutic administration or immunization of the peptides described herein will be apparent to those skilled in the art from the description herein.

[0166] An adjuvant is any substance whose admixture with an immunogenic composition enhances or otherwise modifies the immune response to a therapeutic agent. A carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which a non-mutated protein epitope polypeptide or polynucleotide can associate. Optionally, an adjuvant is conjugated covalently or non-covalently to a polypeptide or polynucleotide described herein.

[0167] The ability of an adjuvant to enhance the immune response to an antigen typically manifests as a marked increase in the immune-mediated reaction or a reduction in disease symptoms. For example, an increase in humoral immunity may manifest as a marked increase in the titer of antibodies generated against an antigen, and an increase in T cell activity may manifest as an increase in cell proliferation or the cytotoxicity or cytokine secretion of cells. In addition, an adjuvant can alter the immune response, for example, by changing a mainly humoral response or a helper T2-type response to a mainly cellular response or a helper T1-type response.

[0168] Suitable adjuvants are known in the art (see WO2015 / 095811) and include poly(I:C), poly-ICLC, STING agonists, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila’s QS21 Stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics and other proprietary adjuvants, such as Ribi’s Detox.Quil or Superfos, but are not limited thereto. Also included as adjuvants are incomplete Freund's adjuvant or GM-CSF. Some immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Dupuis M et al., Cell Immunol. 1998;186(1):18-27; Allison A C; Dev Biol Stand. 1998;92:3-11) (Mosca et al., Frontiers in Bioscience, 20 07;12:4050-4060) (Gamvrellis et al., Immunol & Cell Biol. 20 2004; 82 volumes: 506 - 516 pages). Also, cytokines can be used. Some cytokines affect dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerate the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1b, IL-4, IL-6 and CD40L) (U.S. Patent No. 5,849,589, which is hereby incorporated by reference in its entirety), and act as immunoadjuvants (e.g., IL-12) (Gabrilovich D I et al., J Immunother Emphasis Tumor Immunol. 1996 (6 volumes): 414 - 418 pages) are directly relevant.

[0169] In addition, CpG immunostimulatory oligonucleotides have been reported to enhance the effect of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (nonadaptive) immune system via Toll-like receptors (TLRs), mainly TLR9. CpG-induced TLR9 activation improves antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines, and polysaccharide conjugates of both prophylactic and therapeutic vaccines. Importantly, it improves dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and strong cytotoxic T lymphocyte (CTL) generation even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or coadministered with other adjuvants or when in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations that are particularly necessary to induce strong responses when the antigen is relatively weak. They also accelerate the immune response and have enabled reduction of the antigen dose with an antibody response comparable to that of a full-dose vaccine without CpG in some experiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, Volume 5, June 2006, Pages 471 - 484). U.S. Patent No. 6,406,7 05B1 describes the use of a combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce an antigen-specific immune response. The commercially available CpG TLR9 antagonist, dSLIM (double Stem Loop It is an immunomodulator. Also, other TLR-binding molecules such as RNA-binding TLR7, TLR8, and / or TLR9 may be used.

[0170] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., poly i:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 for TLR8, and bioactive small molecules and antibodies that can act as a treatment and / or as an adjuvant, such as cyclophosphamide, sunitinib, bevacizumab, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD 2171, AZD2171, ipilimumab, tremelimumab, and SC58175, but are not limited thereto. The amounts and concentrations of adjuvants and additives useful in the present invention can be readily determined by those skilled in the art without undue experimentation. As additional adjuvants, colony-stimulating factors such as granulocyte macrophage colony-stimulating factor (GM-CSF, sargramostim) are included.

[0171] In some embodiments, the immunogenic composition according to the present invention may contain more than one various adjuvants. Further, the present invention encompasses a therapeutic composition containing any adjuvant substance including any one or a combination thereof as described above. Also contemplated are non-mutated protein epitope therapeutics (e.g., humoral or cell-mediated immune responses). In some embodiments, the immunogenic composition contains a non-mutated protein epitope therapeutic (e.g., peptide, polynucleotide, TCR, CAR, cell containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.), and the adjuvant can be administered separately in any suitable order.

[0172] The carrier can exist independently of the adjuvant. The functions of the carrier can be, for example, increasing the molecular weight of specific variants to increase their activity or immunogenicity, providing stability, increasing biological activity, or increasing the serum half-life. Furthermore, the carrier can assist in presenting the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. The carrier protein can be keyhole limpet hemocyanin, a serum protein such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, an immunoglobulin or a hormone such as insulin or palmitic acid, but is not limited thereto. In one embodiment, the carrier comprises a human fibronectin type III domain (Koide et al., Methods Enzymol. 2012; 503: 135-56). For human immunization, the carrier must be physiologically acceptable and safe for humans. However, in one embodiment of the present invention, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be dextran, such as sepharose.

[0173] In some embodiments, the polypeptide can be synthesized as a peptide linked in various ways as an alternative to coupling the polypeptide to a carrier to increase immunogenicity. Such molecules are also known as multiple antigen peptides (MAP).

[0174] IV. Combination of CTL Peptide and HTL Peptide An immunogenic composition or vaccine composition comprising a non-mutated protein epitope polypeptide and polynucleotide or an analog thereof described herein having immunostimulatory activity may be modified to provide desired attributes, such as an improved serum half-life, or to enhance immunogenicity.

[0175] For example, the ability of a non-mutated protein epitope peptide to induce CTL activity can be improved by linking the peptide to a sequence containing at least one epitope capable of inducing a helper T cell response. In one embodiment, the CTL epitope / HTL epitope conjugate is linked by a spacer molecule. The spacer typically consists of a relatively small neutral molecule that is substantially uncharged under physiological conditions, such as an amino acid or an amino acid mimetic. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. Optionally, the spacer need not consist of the same residues and thus can be a hetero-oligomer or a homo-oligomer, which is understood. When present, the spacer is usually at least 1 or 2 residues, more usually 3 - 6 residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.

[0176] The CTL peptide epitope may be directly linked to the T helper peptide epitope, or the CTL epitope / HTL epitope conjugate may be linked by a spacer molecule. The spacer typically consists of a relatively small neutral molecule that is substantially uncharged under physiological conditions, such as an amino acid or an amino acid mimetic. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. Optionally, the spacer need not consist of the same residues and thus can be a hetero-oligomer or a homo-oligomer, which is understood. When present, the spacer is usually at least 1 or 2 residues, more usually 3 - 6 residues. The CTL peptide epitope may be directly or via a spacer linked to the T helper peptide epitope at either the amino terminus or the carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

[0177] Also, HTL peptide epitopes may be modified to alter their biological properties. For example, peptides containing HTL epitopes may contain D - amino acids to increase their resistance to proteases and thus extend their serum half - life. Also, epitope peptides may be conjugated to other molecules, such as lipids, proteins or sugars or any other synthetic compound, to increase their biological activity. For example, T - helper peptides may be conjugated to one or more palmitic acid chains at either the amino - terminal or carboxyl - terminal end.

[0178] In certain embodiments, the T - helper peptide is a peptide recognized by helper T cells present in the majority of a population. This can be achieved by selecting amino acid sequences that bind to many, most or all of the HLA class II molecules. These are known as "loosely HLA - restricted" or "promiscuous" T - helper sequences. Examples of promiscuous amino acid sequences include sequences from antigens such as positions 830 - 843 of tetanus toxoid (QYIKANSKFIGITE), positions 378 - 398 of Plasmodium falciparum CS protein (DIEKKIAKMEKASSVFNVVNS) and position 116 of Streptococcus 18kD protein (GAVDSILGGVATYGAA). Other examples include peptides having either the DR1 - 4 - 7 supermotif or the DR3 motif.

[0179] Alternatively, it is possible to prepare synthetic peptides that can stimulate helper T lymphocytes in a mild HLA-restricted manner using amino acid sequences not found in nature (see, for example, PCT Publication WO 95 / 07707). These synthetic compounds, called Pan-DR binding epitopes (e.g., PADRE, Epimmune, Inc., San Diego, CA), are designed to bind to most HLA-DR (human HLA class II) molecules. For example, a Pan-DR binding epitope peptide having the formula: aKXVWANTLKAAa, where "X" is either cyclohexylalanine, phenylalanine or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles and to stimulate the response of helper T lymphocytes from most individuals regardless of HLA type. Alternatives to the Pan-DR binding epitope can contain all natural "L" amino acids and can be provided in the form of nucleic acids encoding the epitope.

[0180] In some embodiments, it may be desirable to include in a pharmaceutical composition (e.g., an immunogenic composition) a non-mutated protein epitope therapeutic agent (e.g., a peptide, polynucleotide, TCR, CAR, cell containing a TCR or CAR, dendritic cell containing a polypeptide, dendritic cell containing a polynucleotide, antibody, etc.) at least one component that pre-stimulates cytotoxic T lymphocytes. Lipids have been identified as agents capable of pre-stimulating CTLs in vivo against viral antigens. For example, palmitic acid residues can bind to the ε-amino and α-amino groups of lysine residues and then be linked to an immunogenic non-mutated protein epitope peptide, for example, via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, etc. The lipidated peptide may then be administered directly as micelles or particles, incorporated into liposomes, or emulsified in an adjuvant. In one embodiment, a particularly effective immunogenic construct comprises palmitic acid bound to the ε-amino and α-amino groups of Lys, which is bound to the amino terminus of the immunogenic peptide via a linkage, for example, Ser-Ser.

[0181] As another example of lipid pre-stimulation of CTL responses, E. coli lipoprotein, such as tripalmitoyl-S-glycerylcysteinylseryl (glycerylcysteinlyseryl)-serine (P3CSS), can be used to pre-stimulate virus-specific CTLs when covalently attached to an appropriate peptide. (See, for example, Deres et al., Nature 342 Vol: 561, page 1989, 1989). The non-mutated protein epitope peptides described herein may, for example, be coupled to P3CSS, and the lipopeptide can be administered to an individual to specifically pre-stimulate a CTL response against a target antigen. Additionally, induction of neutralizing antibodies can be pre-stimulated by epitopes conjugated with P3CSS, so two such compositions may be combined to more effectively induce both humoral and cell-mediated responses against infection.

[0182] As shown herein, additional amino acids may be added to the ends of non-mutated protein epitope peptides to facilitate linking peptides to each other, to provide coupling to a carrier support or a larger peptide, to provide modification of the physical or chemical properties of the peptide or oligopeptide, etc. Amino acids such as tyrosine, cysteine, lysine, glutamic acid or aspartic acid may be introduced at the C-terminus or N-terminus of the peptide or oligopeptide. However, it should be noted that modification at the carboxyl terminus of the T cell epitope may, in some cases, alter the binding properties of the peptide. In addition, the peptide or oligopeptide sequence may be modified by terminal NH2 acylation, such as alkanoyl (C1-C20) or thioglycolyl acetylation, terminal carboxyl amidation, such as by ammonia, methylamine, etc., and thus may differ from the native sequence. In some cases, these modifications may provide a site for linkage to a support or other molecule.

[0183] Embodiments of the immunogenic compositions described herein include ex vivo administration of a cocktail of non-mutated protein epitope polypeptides or polynucleotides having epitopes to PBMCs from a patient's blood or DCs isolated therefrom. Pharmaceuticals for promoting the recovery of dendritic cells (DCs), including GM-CSF, IL-4, IL-6, IL-1β and TNFα, may be used. After pulsing the DCs with the peptide or polynucleotide encoding the peptide and before reinjecting the patient, the DCs are washed to remove unbound peptide. In this embodiment, the vaccine or immunogenic composition comprises DCs pulsed with a peptide that presents a pulsed peptide epitope complexed with an HLA molecule on its surface. The composition is then administered to the patient. In other embodiments, such pulsed DCs are used to stimulate T cells suitable for use in T cell therapy.

[0184] V. Multi-epitope immunogenic compositions Several different approaches are available that enable the simultaneous delivery of multiple epitopes. Nucleic acids encoding non-mutated protein epitope peptides described herein are a particularly useful embodiment of the invention. In one embodiment, the nucleic acid is RNA. In some embodiments, a minigene construct encoding a non-mutated protein epitope peptide comprising one or more epitopes described herein is used to administer a nucleic acid encoding a non-mutated protein epitope peptide described herein.

[0185] The use of multi-epitope minigenes has been described by An, L. and Whitton, J. L., J. Virol. 71 Vol: 2292, 1997; Thomson, S. A. et al., J. Immunol. 157: 822, 1996; Whitton, J. L. et al., J. Virol. 67: 348, 1993; Hanke, R. et al., Vaccine 16: 426, 1998. For example, multi-epitope DNA plasmids encoding antigenic peptides having supermotifs and / or antigenic peptides having motifs, universal helper T cell epitopes (or multiple tumor-associated antigen HTL epitopes), and endoplasmic reticulum translocation signal sequences can be engineered.

[0186] The immunogenicity of multi-epitope minigenes can be tested in transgenic mice to evaluate the magnitude of the immune response induced against the tested epitopes. Furthermore, the immunogenicity of epitopes encoded by DNA in vivo can correlate with the in vitro response of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show both 1.) that the minigene functions to generate a cell-mediated and / or humoral response, and 2.) that the induced immune cells recognize cells expressing the encoded epitope.

[0187] ​For example, to create a DNA sequence (mini - gene) encoding a selected non - mutated protein epitope for expression in human cells, the amino acid sequence of the epitope can be reverse - translated. A human codon usage table can be used to derive codon options for each amino acid. The DNA sequences encoding these non - mutated protein epitopes may be directly adjacent such that when translated, a continuous polypeptide sequence is created. To optimize expression and / or immunogenicity, additional elements may be incorporated into the mini - gene design. Examples of amino acid sequences that can be reverse - translated and included in the mini - gene sequence include HLA class I epitopes, HLA class II epitopes, ubiquitination signal sequences and / or endoplasmic reticulum targeting signal sequences. In addition, HLA presentation of CTL and HTL epitopes can be improved by including synthetic (e.g., polyalanine) or naturally occurring adjacent sequences adjacent to the CTL or HTL epitope, and these larger peptides containing the epitope(s) are within the scope of the present invention.

[0188] The mini - gene sequence can be converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the mini - gene. Overlapping oligonucleotides (30 - 100 bases in length) can be synthesized, phosphorylated, purified, and annealed under appropriate conditions using well - known techniques. The ends of the oligonucleotides may be joined using, for example, T4 DNA ligase. This synthetic mini - gene encoding the epitope polypeptide can then be cloned into a desired expression vector.

[0189] Standard regulatory sequences well known to those skilled in the art can be included in the vector to ensure expression in target cells. For example, a promoter having a downstream cloning site for mini-gene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Many promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. For other suitable promoter sequences, see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466.

[0190] Additional vector modifications can be used to optimize mini-gene expression and immunogenicity. In some cases, introns are utilized for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcribed region of the mini-gene. Also, including mRNA stabilizing sequences and sequences for replication in mammalian cells can be considered to increase mini-gene expression.

[0191] Once an expression vector is selected, the mini-gene can be cloned into the polylinker region downstream of the promoter. An appropriate E. coli strain is transformed with this plasmid, and the DNA is prepared using standard techniques. The orientation and DNA sequence of the mini-gene and all other elements contained in the vector can be confirmed using restriction enzyme mapping and DNA sequence analysis. Bacterial cells having the correct plasmid may be stored as a master cell bank and a working cell bank.

[0192] In addition, immunomodulatory sequences appear to play a role in the immunogenicity of DNA vaccines. These sequences can be included in the vector outside of the mini-gene coding sequence to improve immunogenicity if desired. In one embodiment, the sequence is immunostimulatory. In another embodiment, the sequence is ISS or CpG.

[0193] In some embodiments, a bicistronic expression vector can be used that enables the production of both the epitope encoded by the mini-gene and a second protein (included to enhance or decrease immunogenicity). Examples of proteins or polypeptides that, when co-expressed, can beneficially enhance the immune response include cytokines (such as IL-2, IL-12, GM-CSF), cytokine-inducing molecules (such as LeIF), co-stimulatory molecules, or pan-DR-binding proteins for HTL responses. Helper (HTL) epitopes can be conjugated to an intracellular targeting signal and expressed separately from the CTL epitope that is expressed, which allows for the targeting of the HTL epitope to a different cellular compartment than that of the CTL epitope. If necessary, this can facilitate the more efficient entry of the HTL epitope into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, the co-expression of immunosuppressive molecules (such as TGF-β) to specifically decrease the immune response can be beneficial in certain diseases.

[0194] Therapeutic amounts of plasmid DNA can be produced, for example, by fermentation in E. coli followed by purification. A defined aliquot from a working cell bank is used to inoculate the growth medium and grown to saturation in a shake flask or bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation techniques, such as solid-phase anion exchange resins supplied by QIAGEN, Inc. (Valencia, California). If desired, supercoiled DNA can be isolated from open circular and linear forms using gel electrophoresis or other methods.

[0195] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). This approach, known as "naked DNA", is currently being used in clinical trials for intramuscular (IM) administration. Alternative methods for formulating purified plasmid DNA may be used to maximize the immunotherapeutic effect of mini-gene DNA vaccines. A variety of methods have been described and new technologies may become available. In addition, cationic lipids can be used in formulations (see, for example, WO93 / 24640; Mannino and Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Patent No. 5,279,833; WO91 / 06309; and Felgner et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987)). In addition, compounds collectively referred to as glycolipids, fusogenic liposomes, peptides, and protective, interactive, non-condensing compounds (PINC) can be complexed to purified plasmid DNA to affect variables such as stability, intramuscular dispersion, or transport to specific organs or cell types. See those described by

[0196] In another embodiment, nucleic acids are introduced into cells by use of high-speed cell deformation. During high-speed deformation, temporary disruption of the cell membrane occurs and the cells are thus squeezed so that the nucleic acids can enter the cells. Alternatively, proteins may be produced from an expression vector, for example in a bacterial expression vector, and the proteins can then be delivered to the cells.

[0197] Target cell sensitization can be used as a functional assay for the expression of CTL epitopes encoded by the minigene and HLA class I presentation. For example, plasmid DNA is introduced into mammalian cell lines suitable as targets in a standard CTL chromium release assay. The transfection method used depends on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids enable direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to enable enrichment of transfected cells using fluorescence-activated cell sorting (FACS). These cells are then labeled with chromium-51 ( 51 Cr) and used as target cells for epitope-specific CTL lines. 51 Cytolysis detected by Cr release indicates both the generation of CTL epitopes encoded by the minigene and their HLA presentation. Expression of HTL epitopes can be evaluated in a similar manner using an assay to assess HTL activity.

[0198] In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dosage and route of administration depend on the formulation (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA). An exemplary protocol is at 21 days post-immunization, when splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope to be tested. Subsequently, for CTL effector cells, an assay is performed for cytolysis of peptide-loaded 51 Cr-labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loaded with peptide epitopes corresponding to the epitopes encoded by the minigene indicates the DNA vaccine function for in vivo induction of CTL. Immunogenicity of HTL epitopes is evaluated in transgenic mice in a similar manner.

[0199] Alternatively, the nucleic acid can be administered using, for example, the ballistic delivery described in U.S. Patent No. 5,204,253. Using this technique, particles consisting of only DNA are administered. In a further alternative embodiment, the DNA can be attached to particles, such as gold particles.

[0200] VI. Cells In one aspect, the invention also provides cells that express a non-mutated protein epitope recognition receptor (e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR)) that activates an immune-responsive cell and methods of using such cells for the treatment of diseases that require enhancement of the immune response.

[0201] Such cells include genetically modified immune-responsive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocyte (CTL) cells, helper T lymphocyte (HTL) cells) that express an antigen recognition receptor (e.g., a TCR or a CAR) that binds to one of the non-mutated protein epitope peptides described herein and thus methods of use for the treatment of neoplasms and other lesions where an increase in antigen-specific immune response is desired. T cell activation is mediated by a TCR or a CAR targeted to the antigen.

[0202] The invention provides cells that express a combination of an antigen recognition receptor (e.g., a TCR, a CAR) that activates an immune-responsive cell and a chimeric costimulatory receptor (CCR) and methods of using such cells for the treatment of diseases that require enhancement of the immune response. In one embodiment, tumor antigen-specific T cells, NK cells, CTL cells, or other immune-responsive cells are used as shuttles for the selective enrichment of one or more costimulatory ligands for the treatment or prevention of neoplasms. Such cells are administered to a human subject in need thereof for the treatment or prevention of a particular cancer.

[0203] In one embodiment, tumor antigen-specific human lymphocytes that can be used in the method of the present invention include, but are not limited to, peripheral donor lymphocytes genetically modified to express a chimeric antigen receptor (CAR) (Sadelain, M. et al., 2003, Nat Rev Cancer 3:35-45), a and peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex containing the p heterodimer (Morgan, R. A. et al., 2006, Science 314:126-129), lymphocyte cultures derived from tumor infiltrating lymphocytes (TIL) during tumor biopsy (Panelli, M. C. et al., 2000, J Immunol 164:495-504; Panelli, M. C. et al., 2000, J Immunol 164:4382-4392) and antigen-specific peripheral blood leukocytes selectively expanded and proliferated in vitro using artificial antigen presenting cells (AAPC) or pulsed dendritic cells (Dupont, J. et al., 2005, Cancer Res 65:5417-5427; Papanicolaou, G. A. et al., 2003, Blood 102:2498-2505). The T cells may be autologous, allogeneic, or may be derived in vitro from engineered progenitor cells or stem cells.

[0204] Co-stimulatory ligand In one embodiment, the cells of the present invention are provided with at least one co-stimulatory ligand, which is a non-antigen-specific signal important for the complete activation of immune cells. Co-stimulatory ligands include, but are not limited to, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15 or IL21) and immunoglobulin (Ig) superfamily ligands.

[0205] Tumor necrosis factor (TNF) is a cytokine associated with systemic inflammation and stimulates the acute-phase response. Its main role lies in the regulation of immune cells. Tumor necrosis factor (TNF) ligands share several common features. Most of the ligands are synthesized as type II transmembrane proteins containing short cytoplasmic segments and relatively long extracellular regions. TNF ligands include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, tumor necrosis factor alpha (TNFα), CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B cell-activating factor (BAFF) / Blys / THANK / TALL-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins. They have immunoglobulin domains (folds). Immunoglobulin superfamily ligands include, but are not limited to, both ligands for CD80 and CD86, and ligands for CD28.

[0206] The composition containing the genetically modified immune-responsive cells of the present invention can be provided systemically or directly to a subject for the treatment of a neoplasm. In one embodiment, the cells of the present invention are directly injected into the organ of interest (e.g., the organ affected by a tumor). Alternatively, the composition containing the genetically modified immune-responsive cells is indirectly provided to the organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). To increase the generation of T cells, NK cells, or CTL cells in vitro or in vivo, expansion and differentiation agents may be provided before, during, or after the administration of the cells.

[0207] The modified cells can be administered, usually intravascularly, in any physiologically acceptable vehicle, but they can also be introduced into bone or other convenient sites (such as the thymus) where the cells can find a suitable site for regeneration and differentiation. The genetically modified immune-responsive cells of the present invention can comprise a purified cell population. Those skilled in the art can readily determine the proportion of genetically modified immune-responsive cells in the population using various well-known methods, such as fluorescence-activated cell sorting (FACS). The dosage can be readily adjusted by those skilled in the art (for example, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc. If desired, it may also include factors including, but not limited to, interleukins such as IL-2, IL-3, IL-6 and IL-11 and other interleukins, colony-stimulating factors such as G-, M- and GM-CSF, interferons such as gamma-interferon, and erythropoietin.

[0208] The compositions of the present invention include pharmaceutical compositions comprising genetically modified immune-responsive cells or their progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, the immune-responsive cells or progenitor cells can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived immune-responsive cells of the present invention or their progeny (e.g., derived in vivo, ex vivo or in vitro) can be administered by local injection, systemic injection, topical injection, intravenous injection or parenteral administration, including catheter administration. When administering the therapeutic compositions of the present invention (e.g., pharmaceutical compositions containing genetically modified immune-responsive cells), it is generally formulated in unit dosage injection form (solution, suspension, emulsion).

[0209] VII. Methods of Use and Pharmaceutical Compositions The non-mutated protein epitope therapeutic agents (e.g., peptides, polynucleotides, TCRs, CARs, cells containing TCRs or CARs, dendritic cells containing polypeptides, dendritic cells containing polynucleotides, antibodies, etc.) described in this specification are useful in various applications, including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In some embodiments, the therapeutic treatment method includes immunotherapy. In certain embodiments, the non-mutated protein epitope peptide is useful for activating, promoting, increasing, and / or enhancing the immune response, redirecting the existing immune response to a new target, increasing the immunogenicity of the tumor, inhibiting tumor growth, reducing tumor volume, increasing tumor cell apoptosis, and / or reducing the tumorigenic ability of the tumor. The method of use can be an in vitro, ex vivo, or in vivo method.

[0210] In one aspect, the present invention provides a method for activating an immune response in a subject using a non-mutated protein epitope therapeutic agent described herein. In some embodiments, the present invention provides a method for promoting an immune response in a subject using a non-mutated protein epitope therapeutic agent described herein. In some embodiments, the present invention provides a method for increasing an immune response in a subject using a non-mutated protein epitope peptide described herein. In some embodiments, the present invention provides a method for improving an immune response using a non-mutated protein epitope peptide. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing cell-mediated immunity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing T cell activity or humoral immunity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing CTL or HTL activity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing T cell activity and increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes increasing CTL activity and increasing NK cell activity. In some embodiments, the activation, promotion, increase and / or improvement of the immune response includes inhibiting or decreasing the inhibitory activity of Tregs. In some embodiments, the immune response is a result of an antigenic stimulus. In some embodiments, the antigenic stimulus is a tumor cell. In some embodiments, the antigenic stimulus is cancer.

[0211] In some embodiments, the present invention provides a method of activating, promoting, increasing, and / or enhancing an immune response using the non-mutated protein epitope therapeutics described herein. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope therapeutic that delivers a non-mutated protein epitope polypeptide or polynucleotide to tumor cells. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope therapeutic that binds to a tumor-associated antigen and is internalized by tumor cells. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide that is internalized by tumor cells, wherein the non-mutated protein epitope peptide is processed by the cells. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide that is internalized by tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide that is internalized by tumor cells and processed by the cells, wherein the antigenic peptide is presented on the surface of the tumor cells.

[0212] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide or polynucleotide described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells. In some embodiments, the antigenic peptide is presented on the surface of the tumor cells in a complex with an MHC class I molecule. In some embodiments, the antigenic peptide is presented on the surface of the tumor cells in a complex with an MHC class II molecule.

[0213] In some embodiments, the method comprises contacting tumor cells with a non-mutated protein epitope polypeptide or polynucleotide as described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to the tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells. In some embodiments, the antigenic peptide is presented on the surface of the tumor cells in complex with an MHC class I molecule. In some embodiments, the antigenic peptide is presented on the surface of the tumor cells in complex with an MHC class II molecule.

[0214] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide or polynucleotide as described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to the tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells and an immune response against the tumor cells is induced. In some embodiments, the immune response against the tumor cells is increased. In some embodiments, the non-mutated protein epitope polypeptide or polynucleotide delivers an exogenous polypeptide comprising at least one antigenic peptide to the tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells and tumor growth is inhibited.

[0215] In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a non-mutated protein epitope polypeptide or polynucleotide as described herein that delivers an exogenous polypeptide comprising at least one antigenic peptide to the tumor cells, wherein the antigenic peptide is presented on the surface of the tumor cells and killing by T cells directed against the tumor cells is induced. In some embodiments, the killing by T cells directed against the tumor cells is improved. In some embodiments, the killing by T cells directed against the tumor cells is increased.

[0216] In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a non-mutated protein epitope therapeutic agent described herein, where the agent is an antibody that specifically binds to a non-mutated protein epitope described herein. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of an antibody.

[0217] The present invention provides a method of redirecting an existing immune response back to a tumor. In some embodiments, a method of redirecting an existing immune response back to a tumor comprises administering to the subject a therapeutically effective amount of a non-mutated protein epitope therapeutic agent described herein. In some embodiments, the existing immune response is against a virus. In some embodiments, the virus is selected from the group consisting of measles virus, varicella-zoster virus (VZV; chickenpox virus), influenza virus, mumps virus, poliovirus, rubella virus, rotavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). In some embodiments, the virus is varicella-zoster virus. In some embodiments, the virus is cytomegalovirus. In some embodiments, the virus is measles virus. In some embodiments, the existing immune response has been acquired after a natural viral infection. In some embodiments, the existing immune response has been acquired after vaccination against a virus. In some embodiments, the existing immune response is a cell-mediated response. In some embodiments, the existing immune response comprises cytotoxic T cells (CTLs) or HTLs.

[0218] In some embodiments, a method of redirecting an immune response present in a subject back to a tumor involves administering a fusion protein comprising (i) an antibody that specifically binds to a non-mutated protein epitope and (ii) at least one non-mutated protein epitope peptide described herein, wherein (a) the fusion protein is internalized by tumor cells after binding to a tumor-associated antigen, (b) the non-mutated protein epitope peptide is processed, associates with MHC class I molecules, and is presented on the surface of the tumor cells, and (c) the non-mutated protein epitope peptide / MHC class I complex is recognized by cytotoxic T cells. In some embodiments, the cytotoxic T cells are memory T cells. In some embodiments, the memory T cells are the result of vaccination with the non-mutated protein epitope peptide.

[0219] The present invention provides a method of increasing the immunogenicity of a tumor. In some embodiments, the method of increasing the immunogenicity of a tumor involves contacting the tumor or tumor cells with an effective amount of a non-mutated protein epitope therapeutic agent described herein. In some embodiments, the method of increasing the immunogenicity of a tumor involves administering to a subject a therapeutically effective amount of a non-mutated protein epitope therapeutic agent described herein.

[0220] The present invention also provides a method for inhibiting tumor growth using the non-mutated protein epitope therapeutic agents described herein. In certain embodiments, the method for inhibiting tumor growth comprises contacting a cell mixture with a non-mutated protein epitope therapeutic agent in vitro. For example, an immortalized cell line or cancer cell line mixed with immune cells (e.g., T cells) is cultured in a medium, and a non-mutated protein epitope peptide is added thereto. In some embodiments, tumor cells are isolated from a patient sample, such as a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., T cells), cultured in a medium, and an antigen therapeutic agent is added thereto. In some embodiments, the non-mutated protein epitope therapeutic agent increases, promotes, and / or enhances the activity of immune cells. In some embodiments, the non-mutated protein epitope therapeutic agent inhibits tumor cell growth. In some embodiments, the non-mutated protein epitope therapeutic agent activates the killing of tumor cells.

[0221] In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or the subject had a tumor that was at least partially removed.

[0222] In some embodiments, the method for inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a non-mutated protein epitope therapeutic agent to redirect an existing immune response to a new target, wherein the existing immune response is against an antigenic peptide delivered to tumor cells by a non-mutated protein epitope peptide.

[0223] In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the incidence of cancer stem cells in the tumor is reduced by administration of a non-mutated protein epitope therapeutic agent. In some embodiments, provided is a method for reducing the incidence of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a non-mutated protein epitope therapeutic agent.

[0224] In addition, in some embodiments, the present invention provides a method of reducing the tumorigenic ability of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the non-mutated protein epitope therapeutic agent described herein. In certain embodiments, the tumor comprises cancer stem cells. In some embodiments, the tumorigenic ability of the tumor is reduced by reducing the frequency of occurrence of cancer stem cells in the tumor. In some embodiments, the method comprises using the non-mutated protein epitope therapeutic agent described herein. In certain embodiments, the frequency of occurrence of cancer stem cells in the tumor is reduced by administration of the non-mutated protein epitope therapeutic agent described herein.

[0225] In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of colorectal tumors, pancreatic tumors, lung tumors, ovarian tumors, liver tumors, breast tumors, kidney tumors, prostate tumors, neuroendocrine tumors, gastrointestinal tumors, melanomas, cervical tumors, bladder tumors, glioblastomas, and head and neck tumors. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a breast tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a solid tumor.

[0226] The present invention further provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the non-mutated protein epitope therapeutic agent described herein.

[0227] In some embodiments, the method of treating cancer comprises redirecting an existing immune response to a new target, the method comprising administering to the subject a therapeutically effective amount of the non-mutated protein epitope therapeutic agent, wherein the existing immune response is against an antigenic peptide delivered to cancer cells by the non-mutated protein epitope peptide.

[0228] The present invention provides a method of treating cancer, which includes administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of the non-mutated protein epitope therapeutic agent described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a cancerous tumor. In certain embodiments, the subject has a tumor that has been at least partially removed.

[0229] In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, kidney cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, glioblastoma, triple-negative breast cancer (TNBC), smoldering multiple myeloma (SMM), and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is melanoma. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer includes solid tumors.

[0230] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a cancer selected from the group consisting of acute myeloid leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloid leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

[0231] In some embodiments, the non-mutated protein epitope therapeutic agent is administered as a combination therapy. Combination therapy with two or more therapeutic agents is not essential, but agents that act by different mechanisms of action are used. Combination therapy using agents with different mechanisms of action can result in additive or synergistic effects. Combination therapy allows for lower doses of each agent than those used in monotherapy, thereby reducing the toxic side effects of the agent(s) and / or increasing the therapeutic index. Combination therapy can reduce the likelihood of the development of resistant cancer cells. In some embodiments, the combination therapy includes a therapeutic agent that affects the immune response (e.g., improves or activates the response) and a therapeutic agent that affects the tumor / cancer cells (e.g., inhibits or kills).

[0232] In some embodiments, the combination of the agents described herein with at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and / or side effects of the agent. In some embodiments, the combination therapy results in a decrease in the toxicity and / or side effects of the additional therapeutic agent(s).

[0233] In certain embodiments, in addition to administering the non-mutated protein epitope therapeutic agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent can be administered before, simultaneously with, and / or following the administration of the agent. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0234] As a therapeutic agent that can be administered in combination with the non-mutated protein epitope therapeutic agent described herein, chemotherapeutic agents are included. Thus, in some embodiments, the method or treatment comprises administration of the agents described herein in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with the agent can be carried out before, simultaneously with, or subsequent to administration of chemotherapy. The combination administration can be in a single pharmaceutical formulation or by co-administration using separate formulations or in any order, but generally includes sequential administration within a period such that all the active agents can exert their biological activities simultaneously. The preparation and administration schedule of such chemotherapeutic agents can be used according to the manufacturer's instructions or as empirically determined by a skilled practitioner. Also, the preparation and administration schedule of such chemotherapy is described in The Chemotherapy Source Book, 4th Edition, 2008, edited by M. C. Perry, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0235] Useful classes of chemotherapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (such as platinum complexes, such as cisplatin, mono(platinum), bis(platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, duocarmycin, etoposide, fluoropyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, purine antagonists, puromycin, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids and the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, a mitotic inhibitor, a topoisomerase inhibitor or an angiogenesis inhibitor.

[0236] Chemotherapeutic agents useful in the present invention include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as bus Rufan, Inprosulfan and Piposulfan; Aziridines such as Benzodopa, Carbocone, Meturedopa and Uredopa; Altretamine, Triethylenemelamine, Triethylenephosphoramide, Triethylenethiophosphaoramide and Trimethylolomelamime; Ethylenimine and Methylamelamines containing Nitrogen Mustards such as Chlorambucil, Chloronaphazine, Colophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan, Novembicin, Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard; Nitrosoureas such as Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimustine; Antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine, Bleomycin, Cactinomycin, Calicheamicin Ruamide (triethylenethiophosphaoramide) and Trimethylolomelamime (trime thylolomelamime); Nitrogen Mustards such as Chlorambucil, Chloronaphazine, Colophosphamide, Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan, Novembicin, Phenesterine, Prednimustine, Trofosfamide, Uracil Mustard; Nitrosoureas such as Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimustine; Antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine, Bleomycin, Cactinomycin, Calicheamicin Isin, carabicin, caminomycin, cardinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diacontin; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triacontin; 2,2’,2’’-trichloroethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;Gacitabine; arabinoside (Ara-C); taxoids such as paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine (XELODA); and any pharmaceutically acceptable salts, acids or derivatives thereof, but not limited thereto. Also, as chemotherapeutic agents, antihormonal agents that act to regulate or inhibit the hormonal action on tumors, such as, for example, antiestrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (FARESTON); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and any pharmaceutically acceptable salts, acids or derivatives thereof. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.;

[0237] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that interferes with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Examples of topoisomerase inhibitors include doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26) and irinotecan and any pharmaceutically acceptable salts, acids or derivatives thereof, but not limited thereto. In some embodiments, the additional therapeutic agent is irinotecan.

[0238] In certain embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical substance that has a structure similar to a metabolite used in normal biochemical reactions but is sufficiently different to interfere with one or more normal functions of cells, such as cell division. Examples of antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, sodium methotrexate, ralitrexed, pemetrexed, tegafur, cytarabine, thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the additional therapeutic agent is gemcitabine.

[0239] In certain embodiments, the chemotherapeutic agent is a mitotic inhibitor, including but not limited to, an agent that binds to tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the mitotic inhibitor includes a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the mitotic inhibitor is an inhibitor of kinesin Eg5 or a mitotic kinase, such as Aurora A or Plk1. In certain embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is albumin-bound paclitaxel.

[0240] In some embodiments, the additional therapeutic agent includes an agent such as a small molecule. For example, the treatment may include combination administration of the agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2) and / or VEGF. In some embodiments, the agent is administered in combination with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR) and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent includes an mTOR inhibitor. In another embodiment, the additional therapeutic agent is chemotherapy or another inhibitor that reduces the number of Treg cells. In certain embodiments, the therapeutic agent is cyclophosphamide or an anti-CTLA4 antibody. In another embodiment, the additional therapeutic agent reduces the presence of myeloid-derived suppressor cells. In a further embodiment, the additional therapeutic agent is docetaxel. In another embodiment, the additional therapeutic agent shifts the cells to a helper T1-type response. In a further embodiment, the additional therapeutic agent is ibrutinib. In some embodiments, the additional therapeutic agent includes a biological molecule, such as an antibody. For example, the treatment may include combination administration of the agent of the present invention with an antibody against tumor-associated antigens including, but not limited to, an antibody that binds to EGFR, HER2 / ErbB2 and / or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab or cetuximab (ERBITUX).

[0241]

[0242] ​ In certain embodiments, the additional therapeutic agent comprises a second immunotherapeutic agent. In some embodiments, the additional immunotherapeutic agent includes, but is not limited to, colony stimulating factors, interleukins, antibodies that block immunosuppressive functions (e.g., anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD3 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies), antibodies that enhance immune cell function (e.g., anti-GITR antibodies, anti-OX-40 antibodies, anti-CD40 antibodies or anti-4-1BB antibodies), toll-like receptors (e.g., TLR4, TLR7, TLR9), soluble ligands (e.g., GITRL, GITRL-Fc, OX-40L, OX-40L-Fc, CD40L, CD40L-Fc, 4-1BB ligand or 4-1BB ligand-Fc) or members of the B7 family (e.g., CD80, CD86). In some embodiments, the additional immunotherapeutic agent targets CTLA-4, CD28, CD3, PD-1, PD-L1, TIGIT, GITR, OX-40, CD-40 or 4-1BB.

[0243] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody selected from the group consisting of nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab, MEDI0680, REGN2810, BGB-A317 and PDR001. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody selected from the group consisting of BMS935559 (MDX-1105), atezolizumab (MPDL3280A), durvalumab (MEDI4736) and avelumab (MSB0010718C). In some embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab (YERVOY) and tremelimumab. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from the group consisting of MEDI6469, MEDI0562 and MOXR0916. In some embodiments, the additional therapeutic agent is an anti-4-1BB antibody selected from the group consisting of PF-05082566.

[0244] In some embodiments, the non-mutated protein epitope therapeutic agent can be administered in combination with a biological molecule selected from the group consisting of adrenomedullin (AM), angiopoietin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophin, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PlGF, gamma-IFN, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[0245] In some embodiments, treatment with the non-mutated protein epitope therapeutic agents described herein can be achieved by surgical removal of the tumor, removal of cancer cells, or any other surgical treatment deemed necessary by the treating physician.

[0246] In certain embodiments, treatment includes administration of the non-mutated protein epitope therapeutic agents described herein in combination with radiation therapy. Treatment with the agent can be performed before, simultaneously with, or following administration of radiation therapy. The schedule of administration of such radiation therapy can be determined by a skilled medical practitioner.

[0247] Combination administration can include administration in a single pharmaceutical formulation, or co-administration using separate formulations or in any order, but generally continuous administration within a period such that all active agents can exert their biological activities simultaneously.

[0248] It is understood that the combination of the non-mutated protein epitope therapeutic agent described herein and at least one additional therapeutic agent may be administered in any order or simultaneously. In some embodiments, the agent is administered to a patient who has previously been treated with a second therapeutic agent. In certain other embodiments, the non-mutated protein epitope therapeutic agent and the second therapeutic agent are administered substantially simultaneously or concurrently. For example, the agent may be given to a subject while the subject is undergoing treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, the non-mutated protein epitope therapeutic agent is administered within one year of treatment with the second therapeutic agent. It is further understood that two (or more) agents or treatments may be administered to a subject within approximately a few hours or minutes (i.e., substantially simultaneously).

[0249] For the treatment of a disease, the appropriate dosage of the non-mutated protein epitope therapeutic agent described herein depends on all of the discretion of the treating physician, such as the type of disease being treated, the severity and course of the disease, the responsiveness of the disease, whether the agent is being administered for a therapeutic purpose or a prophylactic purpose, previous treatments, the patient's medical history, etc. The non-mutated protein epitope therapeutic agent can be administered once, or over a series of treatments lasting several days to several months, or until a cure is effected or a reduction in the disease state (e.g., reduction in tumor size) is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body and varies depending on the relative potency of the individual agent. The treating physician can determine the optimal dosage, method of administration, and rate of repetition.

[0250] In some embodiments, the non-mutated protein epitope therapeutic agent may be administered at a first, higher "loading" dose, followed by one or more subsequent, lower doses. In some embodiments, also, the dosing frequency may vary. In some embodiments, the dosing regimen may include administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once a month. For example, the dosing regimen may include an initial loading dose, followed by a weekly maintenance dose, e.g., 1 / 2 of the initial dose. Alternatively, the dosing regimen may include an initial loading dose, followed by a maintenance dose, e.g., 1 / 2 of the initial dose, every other week. Alternatively, the dosing regimen may include three initial doses over three weeks, followed by a maintenance dose of the same amount, e.g., every other week.

[0251] As is known to those of skill in the art, administration of any therapeutic agent can cause side effects and / or toxicity. In some cases, the side effects and / or toxicity are severe enough to preclude administration of a particular agent at a therapeutically effective dose. In some cases, treatment must be discontinued and other agents may be tried. However, many agents in the same therapeutic class exhibit similar side effects and / or toxicity, meaning that patients may have to stop treatment or, if possible, endure the unpleasant side effects associated with the therapeutic agent.

[0252] In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles." In some embodiments, the agent is administered in 3, 4, 5, 6, 7, 8 or more cycles. For example, the agent is administered 6 cycles every two weeks, the agent is administered 6 cycles every three weeks, the agent is administered 4 cycles every two weeks, the agent is administered 4 cycles every three weeks, etc. The dosing schedule may be determined and subsequently modified by those of skill in the art.

[0253] The present invention provides a method of administering to a subject a non-mutated protein epitope therapeutic agent described herein, which includes using an intermittent dosing strategy for administering one or more agents that can reduce side effects and / or toxicity associated with administration of agents such as drugs, chemotherapeutic agents, etc. In some embodiments, a method for treating cancer in a human subject includes administering to the subject a therapeutically effective dose of a non-mutated protein epitope therapeutic agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, a method for treating cancer in a human subject includes administering to the subject a therapeutically effective dose of a non-mutated protein epitope therapeutic agent in combination with a therapeutically effective dose of a second immunotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy includes administering to the subject an initial dose of the non-mutated protein epitope therapeutic agent and subsequent doses of the agent once every about two weeks. In some embodiments, the intermittent dosing strategy includes administering to the subject an initial dose of the non-mutated protein epitope therapeutic agent and subsequent doses of the agent once every about three weeks. In some embodiments, the intermittent dosing strategy includes administering to the subject an initial dose of the non-mutated protein epitope therapeutic agent and subsequent doses of the agent once every about four weeks. In some embodiments, the agent is administered using an intermittent dosing strategy and an additional therapeutic agent is administered weekly.

[0254] The present invention provides a composition comprising a non-mutated protein epitope therapeutic agent described herein. The present invention also provides a pharmaceutical composition comprising a non-mutated protein epitope therapeutic agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical composition finds use in immunotherapy. In some embodiments, the composition finds use in inhibiting tumor growth. In some embodiments, the pharmaceutical composition finds use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the composition finds use in treating cancer. In some embodiments, the pharmaceutical composition finds use in treating cancer in a subject (e.g., a human patient).

[0255] Formulations are prepared for storage and use by combining the antigen therapeutic agent of the invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients and / or stabilizers to be inert components of the formulation or pharmaceutical composition. Exemplary formulations are listed in WO2015 / 095811.

[0256] Suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as TWEEN or polyethylene glycol (PEG) (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London). In one embodiment, the vehicle is 5% dextrose in water.

[0257] The pharmaceutical compositions described herein can be administered in any number of ways for either local or systemic treatment. Administration can be local by epithelial or transdermal patch, ointment, lotion, cream, gel, drops, suppository, spray, liquid and powder; pulmonary by inhalation or insufflation of powder or aerosol including by nebulizer, intratracheal and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intrathecal or intraventricular).

[0258] The therapeutic agent may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in aqueous or non-aqueous media, or suppositories.

[0259] In addition, the non-mutated protein epitope peptides described herein may be encapsulated in microcapsules. Such microcapsules, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, are as described in Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London and are prepared, respectively, by, for example, coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.

[0260] In certain embodiments, the pharmaceutical formulation comprises a non-mutated protein epitope therapeutic agent described herein complexed with liposomes. Methods for generating liposomes are known to those skilled in the art. For example, some liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes may be extruded through a filter of a defined pore size to produce liposomes having a desired diameter.

[0261] In certain embodiments, sustained release preparations comprising the non-mutated protein epitope peptides described herein can be generated. Suitable examples of sustained release preparations include semi-permeable matrices of solid hydrophobic polymers containing a drug, where the matrix is in the form of a shaped article (e.g., a film or microcapsule). Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0262] VIII. Kit The non-mutated protein epitope therapeutic agents described herein may be provided in kit form together with instructions for administration. Typically, the kit may include the desired antigen therapeutic agent in unit dosage form in a container, and instructions for administration. Additionally, additional therapeutic agents, such as cytokines, lymphokines, checkpoint inhibitors, antibodies, may be included in the kit. Also, other kit components that may be desirable, such as sterile syringes, booster doses, and other desired excipients, are included.

[0263] The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be varied or modified to obtain alternative embodiments that comply with the present invention. All patents, patent applications, and publications listed herein are hereby incorporated by reference in their entirety.

Example

[0264] (Example 1) Identification of Mutated Sequences with Potential Immunogenicity The applicants discovered that the following epitopes recur in cancer patients. [Table 1] For each epitope, the full-length amino acid sequence of the non-mutated protein epitope was obtained. Any component 9-mer or 10-mer not found in the germline protein sequence was marked, and using an available algorithm, the binding potential for six common HLA alleles (HLA-A01:01, HLA-A02:01, HLA-A03:01, HLA-A24:02, HLA-B07:02, and HLA-B08:01) was scored. Any peptide with a score better than 1000 nM was designated. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] For each epitope, the full-length amino acid sequence of the non-mutated protein epitope was obtained. Any component 9-mer or 10-mer not found in the germline protein sequence was marked, and using an available algorithm, the binding potential for six common HLA alleles (HLA-A01:01, HLA-A02:01, HLA-A03:01, HLA-A24:02, HLA-B07:02, and HLA-B08:01) was scored. Any peptide with a score better than 1000 nM was designated.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 5-1

Table 5-2

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

[0265] (Example 2) HLA Class I and Class II Binding Assay The following examples of peptide binding to HLA molecules demonstrate the quantification of the binding affinities of HLA class I and class II peptides. The binding assays can be performed with either peptides that have a motif or peptides that do not have a motif.

[0266] An Epstein-Barr virus (EBV)-transformed homozygous cell line, fibroblast, CIR or 721.22 transfectant is used as a source of HLA class I molecules. Cell lysates are prepared and HLA molecules are purified according to the disclosed protocol (Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); Sette et al., Mol. Immunol. 31:8 13 (1994)). HLA molecules are purified from the lysates by affinity chromatography. The lysates are passed through a column of Sepharose CL-4B beads conjugated with the appropriate antibody. The anti-HLA column is then washed with PBS containing 1% NP-40, 10 mM Tris-HCL, pH 8.0 in PBS and 0.4% n-octyl glucoside, and HLA molecules are eluted with 50 mM diethylamine, pH 11.5 in 0.15 M NaCl containing 0.4% n-octyl glucoside. 1 / 25 volume of 2.0 M Tris, pH 6.8 is added to the eluate to reduce the pH to about 8.0. The eluate is then concentrated by centrifugation in a Centriprep 30 concentrator (Amicon, Beverly, MA). The protein content is evaluated by the BCA protein assay (Pierce Chemical Co., Rockford, IL) and confirmed by SDS-PAGE.

[0267] A detailed description of the protocol utilized to measure peptide binding to class I and class II MHCs has been published (Sette et al., Mol. Immunol. 31:8 Page 13, 1994; Sidney et al., Current Protocols in Immunology, edited by Margulies, John Wiley & Sons, New York, Section 18.3, 1998). Briefly, purified MHC molecules (5 - 500 nM) are incubated for 48 hours (or 20% w / v digitonin for the H-2 IA assay) with various unlabeled peptide inhibitors and 1 - 10 nM of 125I radiolabeled probe peptide in PBS containing 0.05% Nonidet P-40 (NP40) in the presence of a protease inhibitor cocktail. DRB1 assays are performed at pH 4.5 * 0301, and DRB1 assays performed at pH 5.0 * 1601 (DR2w21β1) and DRB4 * 0101 (DRw53) are performed at pH 7.0. All other assays are performed at pH 7.0

[0268] After incubation, MHC-peptide complexes are separated from free peptides by gel filtration on a 7.8 mm × 15 cm TSK200 column (TosoHaas 16215, Montgomeryville, PA). Due to the large size of the radiolabeled peptides used in the DRB1 * 1501 (DR2w2β1) assay, it is more difficult to separate the bound peak from the unbound peak under these conditions, so all DRB1 * 1501 (DR2w2β1) assays are performed using a 7.8 mm × 30 cm TSK2000 column and eluted at 0.6 mLs / min. The eluate from the TSK column is passed through a Beckman 170 radioisotope detector, radioactivity is plotted, and integrated using a Hewlett-Packard 3396A integrator to determine the percentage of bound peptide.

[0269] Use the chloramine-T method to iodinate the radiolabeled peptide. Typically, in preliminary experiments, each MHC preparation is titrated in the presence of a fixed amount of radiolabeled peptide to determine the concentration of HLA molecules required to bind 10 - 20% of the total radioactivity. All subsequent inhibition assays and direct binding assays are performed using these HLA concentrations.

[0270] Under these conditions, [label] < [HLA] and IC 50 ≥ [HLA], so the measured IC 50 values are a reasonable approximation of the true K D values. Peptide inhibitors are typically tested at concentrations in the range of 120 μg / ml to 1.2 ng / ml and are tested in 2 - 4 completely independent experiments. To enable comparison of data obtained in different experiments, the IC 50 of the positive control for inhibition is divided by the IC 50 for each peptide tested (typically the unlabeled variant of the radiolabeled probe peptide) to calculate the relative binding number for each peptide. The relative binding values are edited for database purposes and for comparison between experiments. These values can subsequently be converted back to IC 50 nM values by dividing the IC 50 nM of the positive control for inhibition by the relative binding of the peptide of interest. This method of data editing has been shown to be the most accurate and consistent for comparing peptides tested on different days or peptides tested with different lots of purified MHC.

[0271] The antibody (LB3.1) used for HLA - DR purification is α - chain specific, so the β1 molecule is not separated from the β3 (and / or β4 and β5) molecules. The β1 specificity of the binding assay is evident in the cases of DRB1 * 0101 (DR1), DRB1 * 0802 (DR8w2) and DRB1 * 0803 (DR8w3). Also, it is evident in the cases of DRB1 * 0301 (DR3) and DRB3 * 0101 (DR52a), DRB1* 0401 (DR4w4), DRB1 * 0404 (DR4w14), DRB1 * 0405 (DR4w15), DRB1 * 1101 (DR5), DRB1 * 1201 (DR5w12), DRB1 * 1302 (DR6w19) and DRB1 * 0701 (DR7) is also shown. DRB1 * 1501 (DR2w2β1), DRB5 * 0101 (DR2w2β2), DRB1 * 1601 (DR2w21β1), DRB5 * 0201 (DR51Dw21) and DRB4 * The problem of β-chain specificity for the 0101 (DRw53) assay is circumvented by the use of fibroblasts. The development and validation of assays for DRβ molecule specificity have been previously described (see, e.g., Southwood et al., J. Immunol. 16 Vol. 0: pp. 3363 - 3373, 1998).

[0272] Also, a viable cell / flow cytometry-based assay may be used. This is a well-established assay that utilizes the TAP-deficient hybridoma cell line T2 (American Type Culture Collection (ATCC Accession No. CRL-1992), Manassas, Va.). The TAP deficiency in this cell line results in inefficient loading of MHC I in the ER and an excess of empty MHC I. Salter and Cresswell, EMBO J. 5 Vol.: 94 3 - 49 (1986); Salter, Immunogenetics 21 Vol.: 235 - 46 (1985). Empty MHC I is very unstable and, therefore, short-lived. When T2 cells are cultured at low temperature, empty MHC I transiently appears on the cell surface where they can be stabilized by the addition of MHC I-binding peptides from the outside. To perform this binding assay, peptide-receptive MHC I is added in a fixed amount of 10 7Individual T2 cells were induced by culturing overnight at 26°C in serum-free AIM-V medium alone or in medium containing increasing concentrations (0.1 - 100 μM) of the peptide. The cells were then washed twice with PBS and subsequently incubated with the fluorescent-tagged HLA-A0201-specific monoclonal antibody, BB7.2, to quantify cell surface expression. Samples were obtained using a FACS Calibur instrument (Becton Dickinson), and the mean fluorescence intensity (MFI) was determined using the accompanying Cellquest software.

[0273] (Example 3) Confirmation of immunogenicity The in vitro education (IVE) assay is used to test the ability of each test peptide to expand and proliferate CD8+ T cells. Mature professional APCs are prepared for these assays by the following method. 80 - 90 × 10 6 isolated PBMCs from healthy human donors are seeded into 20 ml of RPMI medium containing 2% human AB serum and incubated at 37°C for 2 hours to allow plastic adhesion by monocytes. Non-adherent cells are removed, and the adherent cells are cultured in RPMI, 2% human AB serum, 800 IU / ml of GM-CSF, and 500 IU / ml of IL-4. After 6 days, TNF-alpha is added to a final concentration of 10 ng / ml. On day 7, dendritic cells (DCs) are matured by the addition of 12.5 μg / ml of poly I:C or 0.3 μg / ml of CD40L. Mature dendritic cells (mDCs) are collected on day 8, washed, and used directly or cryopreserved for future use.

[0274] For the IVE of CD8+ T cells, an aliquot of 2 × 10 5 mDCs is pulsed with each peptide at a final concentration of 100 μM, incubated at 37°C for 4 hours, and then irradiated (2500 rad). The peptide-pulsed mDCs are washed twice in RPMI containing 2% human AB serum. 2 × 10 5 mDCs per well and 2 × 10 6Individual autologous CD8+ cells are seeded into 24-well plates in 2 ml of RPMI containing 2% human AB, 20 ng / ml of IL-7 and 100 pg / ml of IL-12 and incubated for 12 days. The CD8+ T cells are then pulsed with peptide and restimulated with irradiated mDC. After 2 to 3 days, 20 IU / ml of IL-2 and 20 ng / IL7 are added. The expanding CD8+ T cells are restimulated every 8 - 10 days and maintained in medium containing IL-2 and IL-7. The cultures are monitored for peptide-specific T cells using a combination of functional assays and / or tetramer staining. Parallel IVE using modified and parental peptides allows comparison of the relative efficiency by which peptides expand peptide-specific T cells.

[0275] Quantitative and functional evaluation of CD8+ T cells Tetramer staining MHC tetramers are purchased or produced in-house and used to measure peptide-specific T cell expansion in the IVE assay. For the assay, according to the manufacturer's instructions, 1×10 5 cells in PBS (FACS buffer) containing 1% FCS and 0.1% sodium azide are added to the tetramers. The cells are incubated for 20 minutes at room temperature in the dark. Antibodies specific for T cell markers, such as CD8, are then added to the final concentration suggested by the manufacturer and the cells are incubated for 20 minutes at 4°C in the dark. The cells are washed with cold FACS buffer and resuspended in buffer containing 1% formaldehyde. The cells are obtained on a FACS Calibur (Becton Dickinson) instrument and analyzed using Cellquest software (Becton Dickinson). For the analysis of tetramer-positive cells, lymphocyte gates are taken from the forward scatter and side scatter plots. The data are reported as the percentage of cells that were CD8+ / tetramer+.

[0276] ELISPOT Functional enumeration of peptide-specific T cells is performed using an ELISPOT assay (BD Biosciences) that measures the release of IFN-γ from T cells on a single cell basis. Target cells (T2 or C1R transfected with HLA-A0201) are pulsed with 10 μM peptide for 1 hour at 37 °C and washed three times. Targets pulsed with 1×10 5 peptides are co-cultured in ELISPOT plate wells with various concentrations of T cells (5×10 2 to 2×103) taken from the IVE culture. The plates are developed according to the manufacturer's protocol and analyzed using the attached software on an ELISPOT reader (Cellular Technology Ltd.). Spots corresponding to the number of IFN-γ-producing T cells are reported as the absolute number of spots per number of seeded T cells. T cells expanded in response to the modified peptide 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 parental peptide.

[0277] CD107 staining CD107a and b are stained on CD8 +It is expressed on the cell surface of T cells. The lytic granules of T cells have a lipid bilayer containing lysosomal-associated membrane glycoproteins (LAMP: lysosomal-associated membrane glycoprotein) including 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 cell membrane of the T cell. The granule contents are released, which causes the death of the target cell. When the granule membrane fuses with the cell membrane, C107a and b are exposed on the cell surface, and thus they are markers of degranulation. Since degranulation 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, peptides were added to C1R cells transfected with HLA-A0201 to a final concentration of 20 μM, the cells were incubated at 37 °C for 1 hour, and washed three times. 1×10 5 C1R cells pulsed with the peptide were aliquoted into tubes, and antibodies specific for CD107a and b were added to the final concentration suggested by the manufacturer (Becton Dickinson). The antibodies are added prior to the addition of T cells to "capture" the CD107 molecules when they transiently appear on the surface during the course of the assay. 1×10 5 T cells from the IVE culture were then added, and the samples were incubated at 37 °C for 4 hours. The T cells were further stained for additional cell surface molecules, such as CD8, and obtained using a FACS Calibur instrument (Becton Dickinson). The data were analyzed using the accompanying Cellquest software, and the results were reported as the percentage of CD8 + CD107a and b + cells.

[0278] CTL lysis Cytotoxic activity is measured using a chromium release assay. Target T2 cells were labeled with Na 51 Cr at 37 °C for 1 hour, washed, and 5×10 3The individual target T2 cells were then added to various numbers of T cells from the IVE culture. Chromium release was measured in the supernatant recovered after 4 hours of incubation at 37°C. The percentage of specific lysis was calculated as: (experimental release - spontaneous release) / (total release - spontaneous release) × 100 as follows.

[0279] (Example 4) Selection of CTL and HTL epitopes for inclusion in tumor - specific vaccines This example illustrates a procedure for selecting peptide epitopes for the vaccine compositions of the present invention. The peptides in the composition may be in the form of a nucleic acid sequence of a single sequence or one or more sequences (i.e., minigenes) encoding the peptide(s), or may be single - epitope peptides and / or multi - epitope peptides.

[0280] Upon administration, epitopes that mimic an immune response that has been observed to correlate with tumor clearance are selected. For example, the vaccine may contain 1 - 2 epitopes derived from at least one tumor antigen region. Epitopes from one region may be used in combination with epitopes from one or more additional tumor antigen regions.

[0281] Epitopes with a binding affinity of 500 nM or less for HLA class I molecules 50 or 1000 nM or less for class II 50 may be selected.

[0282] When creating multi-epitope compositions, such as minigenes, it is typically desirable to generate the smallest possible peptides that contain the epitopes of interest. The principle used is not the same as, but is similar to, the principle used when selecting peptides containing nested epitopes. However, in addition, when determining the nucleic acid sequence provided as a minigene, the peptide sequence it encodes is analyzed to determine whether any "junction epitopes" have been created. Junction epitopes are potential HLA-binding epitopes, predicted, for example, by motif analysis. Since the recipient can bind to the HLA molecule and generate an immune response against that epitope that is not present in the native protein sequence, junction epitopes should generally be avoided.

[0283] Peptide epitopes for inclusion in vaccine compositions are selected, for example, from those listed in the table. A vaccine composition consisting of the selected peptides, when administered, is safe, effective, and induces an immune response of a similar magnitude to that which inhibits tumor growth.

[0284] (Example 5) Peptide compositions for prophylactic or therapeutic use The immunogenic or vaccine composition of the present invention is used to inhibit tumor growth. For example, a multi-epitope composition (or nucleic acid containing the same) containing a plurality of CTL and HTL epitopes is administered to an individual having a tumor. The composition is provided as a single lipidated polypeptide containing a plurality of epitopes. The composition is administered in an aqueous carrier containing alum. The peptide dose for the primary immunization is about 1 to about 50,000 μg, generally 100 - 5,000 μg, for a 70 kg patient. After the primary administration, a booster dose is administered at 4 weeks, and subsequently, the magnitude of the immune response in the patient is evaluated by a technique for determining the presence of an epitope-specific CTL population in the PBMC sample. Additional booster doses are administered as required. The composition has been found to be safe and effective for inhibiting tumor growth.

[0285] Alternatively, the multi-epitope composition may be administered as a nucleic acid, e.g., as RNA, according to methods known in the art and disclosed herein.

[0286] Non-mutated protein epitope binders, e.g., TCRs or CARs, can be administered according to methods known in the art and disclosed herein. The binder may be administered as a polypeptide or polynucleotide, e.g., as RNA encoding the binder, or as a cell therapy by administering cells that express the binder.

[0287] Non-mutated protein epitope peptides, polynucleotides, binders, or cells that express these molecules can be delivered to the same patient by multiple methods known in the art and can be further combined with other cancer treatments (e.g., chemotherapy, surgery, radiation, checkpoint inhibitors, etc.).

[0288] (Example 6) Administration of a composition using dendritic cells The vaccine comprising the epitope of the present invention may be administered using dendritic cells. In this example, dendritic cells pulsed with the peptide can be administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising the peptide CTL and HTL epitopes of the present invention. The dendritic cells are then injected back into the patient to induce CTL and HTL responses in vivo. The induced CTLs and HTLs then destroy (CTL) or promote the destruction of (HTL) specific target tumor cells having the protein from which the epitopes in the vaccine are derived.

[0289] Alternatively, an ex vivo CTL or HTL response against a specific tumor-associated antigen can be induced by incubating the patient's, or genetically compatible, CTL or HTL progenitor cells in tissue culture with an antigen-presenting cell source, such as dendritic cells, and an appropriate immunogenic peptide.

[0290] After an appropriate incubation time (typically about 7 to 28 days) for the progenitor cells to be activated and expanded into effector cells, the cells are injected back into the patient, where they destroy (CTL) or promote the destruction of (HTL) their specific target cells, i.e., tumor cells.

[0291] Paragraph of embodiments An isolated antigenic peptide comprising an epitope derived from the sequence in Table 1 or Table 2.

[0292] An isolated antigenic peptide 100 amino acids in length or less comprising an epitope derived from the sequence in Table 1 or Table 2.

[0293] An isolated antigenic peptide comprising an epitope derived from the sequence in Table 3 or Table 4.

[0294] An isolated antigenic peptide 100 amino acids in length or less comprising an epitope derived from the sequence in Table 3 or Table 4.

[0295] An isolated antigenic peptide comprising an epitope derived from the sequence in Table 5 or Table 6.

[0296] An isolated antigenic peptide 100 amino acids in length or less comprising an epitope derived from the sequence in Table 5 or Table 6.

[0297] An isolated antigenic peptide as described in paragraph

[0291] or

[0292] that is a retroviral antigen.

[0298] An isolated antigenic peptide as described in paragraph

[0293] or

[0294] that is a non-mutated overexpressed antigen.

[0299] An isolated antigenic peptide as described in paragraph

[0295] or

[0296] that is a viral antigen.

[0300] An isolated antigenic peptide as described in any of paragraphs

[0291] to

[0299] that is between about 5 and about 50 amino acids in length.

[0301] An isolated antigenic peptide as described in any of paragraphs

[0291] to

[0300] that is between about 15 and about 35 amino acids in length.

[0302] An isolated antigenic peptide as described in paragraph

[0301] that is about 15 amino acids in length or less.

[0303] An isolated antigenic peptide as described in paragraph

[0302] that is between about 8 and about 11 amino acids in length.

[0304] An isolated antigenic peptide as described in paragraph

[0303] that is 9 or 10 amino acids in length.

[0305] An isolated antigenic peptide as described in any of paragraphs

[0291] to

[0304] that binds to major histocompatibility complex (MHC) class I.

[0306] An isolated antigenic peptide as described in paragraph

[0305] that binds to MHC class I with a binding affinity of less than about 500 nM.

[0307] An isolated antigenic peptide as described in any of paragraphs

[0291] to

[0296] that is about 30 amino acids in length or less.

[0308] An isolated antigenic peptide as described in paragraph

[0307] that is between about 6 and about 25 amino acids in length.

[0309] An isolated antigenic peptide as described in paragraph

[0308] that is between about 15 and about 24 amino acids in length.

[0310] The isolated antigenic peptide described in paragraph

[0308] , which is between about 9 and about 15 amino acids in length.

[0311] The isolated antigenic peptide described in any of paragraphs

[0291] -

[0296] and

[0307] -

[0310] , which binds to MHC class II.

[0312] The isolated antigenic peptide described in paragraph

[0311] , which binds to MHC class II with a binding affinity of less than about 1000 nM.

[0313] The isolated antigenic peptide described in any of paragraphs

[0291] -

[0312] , which further comprises adjacent amino acids.

[0314] The isolated antigenic peptide described in paragraph

[0313] , wherein the adjacent amino acids are not natural adjacent amino acids.

[0315] The isolated antigenic peptide described in any of paragraphs

[0291] -

[0314] , which is linked to at least a second antigenic peptide.

[0316] The isolated antigenic peptide described in paragraph

[0315] , wherein the peptide is linked using a polyglycine or polyserine linker.

[0317] The isolated antigenic peptide described in paragraph

[0315] or

[0316] , wherein the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM.

[0318] The isolated antigenic peptide described in paragraph

[0317] , wherein the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 500 nM.

[0319] The isolated antigenic peptide described in paragraph

[0317] or

[0318] , wherein both of the epitopes bind to human leukocyte antigen (HLA)-A, -B, -C, -DP, -DQ, or -DR.

[0320] The isolated antigenic peptide according to any one of paragraphs

[0317] to

[0319] , wherein the isolated antigenic peptide binds to class I HLA and the second antigenic peptide binds to class II HLA.

[0321] The isolated antigenic peptide according to any one of paragraphs

[0317] to

[0319] , wherein the isolated antigenic peptide binds to class II HLA and the second antigenic peptide binds to class I HLA.

[0322] The isolated antigenic peptide according to any one of paragraphs

[0291] to

[0321] , further comprising a modification that increases in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.

[0323] The isolated antigenic peptide according to paragraph

[0322] , wherein the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polycylation, HESylation, recombinant PEG mimetic, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of an amino acid mimic, or addition of a non-natural amino acid.

[0324] The isolated antigenic peptide according to paragraph

[0322] , wherein the cell to be targeted is an antigen-presenting cell.

[0325] The isolated antigenic peptide according to paragraph

[0324] , wherein the antigen-presenting cell is a dendritic cell.

[0326] The isolated antigenic peptide according to paragraph

[0325] , wherein the dendritic cell is targeted using DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, the TSLP receptor, or the CD1a marker.

[0327] The isolated antigenic peptide according to paragraph

[0326] , wherein the dendritic cell is targeted using the CD141, DEC205, or XCR1 marker.

[0328] An in vivo delivery system comprising the isolated antigenic peptide according to any one of paragraphs

[0291] to

[0327] .

[0329] The delivery system according to paragraph

[0328] , comprising a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, or liposomes.

[0330] The delivery system according to paragraph

[0328] , wherein the cell-penetrating peptide is a TAT peptide, herpes simplex virus VP22, transportan, or Antp.

[0331] A cell comprising the isolated antigenic peptide according to any one of paragraphs

[0291] to

[0327] .

[0332] The cell according to paragraph

[0331] , which is an antigen-presenting cell.

[0333] The cell according to paragraph

[0332] , which is a dendritic cell.

[0334] A composition comprising the isolated antigenic peptide according to any one of paragraphs

[0291] to

[0327] .

[0335] The composition according to paragraph

[0334] , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides comprising a tumor-specific epitope defined in Table 1 or Table 2.

[0336] The composition according to paragraph

[0334] , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides comprising a tumor-specific epitope defined in Table 3 or Table 4.

[0337] The composition according to paragraph

[0334] , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated antigenic peptides, which contain tumor-specific epitopes defined in Table 5 or Table 6.

[0338] The composition according to any one of paragraphs

[0335] to

[0337] , comprising antigenic peptides between 2 and 20.

[0339] The composition according to any one of paragraphs

[0334] to

[0338] , further comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 additional antigenic peptides.

[0340] The composition according to paragraph

[0339] , comprising additional antigenic peptides between about 4 and about 20.

[0341] The composition according to any one of paragraphs

[0334] to

[0340] , wherein the additional antigenic peptides are specific to the tumor of an individual patient.

[0342] The composition according to paragraph

[0341] , wherein the patient-specific antigenic peptide is selected by identifying sequence differences between the genome, exome, and / or transcriptome of the patient's tumor sample and the genome, exome, and / or transcriptome of a non-tumor sample.

[0343] The composition according to paragraph

[0337] , wherein the sample is fresh or formalin-fixed paraffin-embedded tumor tissue, freshly isolated cells, or circulating tumor cells.

[0344] The composition according to paragraph

[0342] or

[0343] , wherein the sequence difference is determined by next-generation sequencing.

[0345] An isolated polynucleotide encoding the isolated antigenic peptide according to any one of paragraphs

[0291] to

[0300] .

[0346] The isolated polynucleotide according to paragraph

[0345] , which is RNA, and optionally self-amplifying RNA.

[0347] The isolated polynucleotide according to paragraph

[0346] , wherein the RNA is modified to increase stability, increase cell targeting, increase translation efficiency, adjuvant activity, cytosolic accessibility, and / or decrease cytotoxicity.

[0348] The isolated polynucleotide according to paragraph

[0347] , wherein the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increase in GC content, incorporation of modified nucleosides, incorporation of a 5'-cap or cap analog, and / or incorporation of an unmasked polyA sequence.

[0349] A cell comprising the polynucleotide according to any one of paragraphs

[0345] to

[0348] .

[0350] A vector comprising the polynucleotide according to any one of paragraphs

[0345] to

[0348] .

[0351] The vector according to paragraph

[0350] , wherein the polynucleotide is operably linked to a promoter.

[0352] The vector according to paragraph

[0350] or

[0351] , which is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion.

[0353] The vector according to paragraph

[0352] , which is an adeno-associated virus, herpes virus, lentivirus, or pseudotype thereof.

[0354] An in vivo delivery system comprising the isolated polynucleotide according to any one of paragraphs

[0345] to

[0348] .

[0355] The delivery system according to paragraph

[0350] , which comprises spherical nucleic acid, virus, virus-like particle, plasmid, bacterial plasmid, or nanoparticle.

[0356] A cell comprising the vector or delivery system according to any one of paragraphs

[0350] to

[0355] .

[0357] The cell according to paragraph

[0356] , which is an antigen-presenting cell.

[0358] The cell according to paragraph

[0357] , which is a dendritic cell.

[0359] The cell according to paragraph

[0358] , which is an immature dendritic cell.

[0360] A composition comprising at least one polynucleotide according to any one of paragraphs

[0345] to

[0348] .

[0361] The composition according to paragraph

[0360] , comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 isolated polynucleotides.

[0362] The composition according to paragraph

[0361] , comprising a polynucleotide between about 2 and about 20.

[0363] The composition according to any one of paragraphs

[0360] to

[0362] , further comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 additional antigenic polynucleotides encoding additional antigenic peptides.

[0364] The composition according to paragraph

[0363] , comprising additional antigenic polynucleotides between about 4 and about 20.

[0365] The composition according to paragraph

[0363] , wherein the isolated polynucleotide and the additional antigenic polynucleotide are linked.

[0366] The composition according to paragraph

[0365] , wherein the polynucleotide is ligated using a nucleic acid encoding a polyglycine or polyserine linker.

[0367] The composition according to any one of paragraphs

[0360] to

[0366] , wherein at least one of the additional antigenic peptides is specific for the tumor of an individual patient.

[0368] The composition according to paragraph

[0367] , wherein the patient-specific antigenic peptide is selected by identifying sequence differences between the genome, exome, and / or transcriptome of the patient's tumor sample and the genome, exome, and / or transcriptome of a non-tumor sample.

[0369] The composition according to paragraph

[0368] , wherein the sample is fresh or formalin-fixed paraffin-embedded tumor tissue, freshly isolated cells, or circulating tumor cells.

[0370] The composition according to paragraph

[0368] or

[0369] , wherein the sequence difference is determined by next-generation sequencing.

[0371] A T cell receptor (TCR) capable of binding to at least one antigenic peptide listed in any one of paragraphs

[0291] to

[0324] .

[0372] The TCR according to paragraph

[0371] , capable of binding to the antigenic peptide isolated in the context of MHC class I or class II.

[0373] A chimeric antigen receptor comprising (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding to an isolated antigenic peptide described in any one of paragraphs

[0291] to

[0324] .

[0374] The chimeric antigen receptor according to paragraph

[0373] , wherein CD3 zeta is the T cell activation molecule.

[0375] The chimeric antigen receptor according to paragraph

[0373] or

[0374] , further comprising at least one co-stimulatory signaling domain.

[0376] The chimeric antigen receptor according to any one of paragraphs

[0373] to

[0375] , wherein the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI gamma.

[0377] The chimeric antigen receptor according to any one of paragraphs

[0373] to

[0376] , wherein the antigen recognition portion can bind to the antigenic peptide isolated in association with MHC class I or class II.

[0378] The chimeric antigen receptor according to any one of paragraphs

[0373] to

[0377] , comprising a transmembrane region of CD3 zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1.

[0379] The chimeric antigen receptor according to any one of paragraphs

[0373] to

[0378] , wherein the tumor-specific epitope is located in the extracellular domain of a tumor-associated polypeptide.

[0380] A T cell comprising a T cell receptor or a chimeric antigen receptor according to any one of paragraphs

[0371] to

[0379] .

[0381] The T cell according to paragraph

[0380] , which is a helper T cell or a cytotoxic T cell.

[0382] A nucleic acid comprising a promoter operably linked to a polynucleotide encoding a T cell receptor according to paragraph

[0371] or

[0372] .

[0383] The nucleic acid according to paragraph

[0382] , wherein the TCR can bind to the at least one antigenic peptide in association with major histocompatibility complex (MHC) class I or class II.

[0384] A nucleic acid comprising a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor as described in any of paragraphs

[0373] to

[0379] .

[0385] The nucleic acid according to paragraph

[0384] , wherein the antigen recognition portion can bind to the at least one antigenic peptide in the context of major histocompatibility complex (MHC) class I or class II.

[0386] The nucleic acid according to paragraph

[0384] or

[0385] , wherein the tumor-specific epitope is located in the extracellular domain of a tumor-associated polypeptide.

[0387] The nucleic acid according to any of paragraphs

[0384] to

[0386] , comprising the transmembrane region of CD3 zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1.

[0388] An antibody capable of binding to at least one antigenic peptide listed in Table 1 or Table 2.

[0389] An antibody capable of binding to at least one antigenic peptide listed in Table 3 or Table 4.

[0390] An antibody capable of binding to at least one antigenic peptide listed in Table 5 or Table 6.

[0391] The antibody according to paragraph

[0388] , wherein at least one antigenic peptide listed in Table 1 or Table 2 is a retroviral antigenic peptide.

[0392] The antibody according to paragraph

[0389] , wherein at least one antigenic peptide listed in Table 3 or Table 4 is a non-mutated overexpressed antigenic peptide.

[0393] The antibody according to paragraph

[0390] , wherein at least one antigenic peptide listed in Table 5 or Table 6 is a viral antigenic peptide.

[0394] A modified cell transfected or transduced with the nucleic acid according to any one of paragraphs

[0382] to

[0387] .

[0395] The modified cell according to paragraph

[0394] , which is a T cell, tumor infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4+ T cell, CD8+ T cell, or NK cell.

[0396] A composition comprising a T cell receptor or chimeric antigen receptor according to any one of paragraphs

[0371] to

[0379] .

[0397] A composition comprising autologous patient T cells containing a T cell receptor or chimeric antigen receptor according to any one of paragraphs

[0371] to

[0379] .

[0398] The composition according to paragraph

[0395] or

[0396] , further comprising an immune checkpoint inhibitor.

[0399] The composition according to paragraph

[0396] or

[0397] , further comprising at least two immune checkpoint inhibitors.

[0400] The composition according to paragraph

[0398] or

[0399] , wherein the immune checkpoint inhibitors each inhibit a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

[0401] The composition according to paragraph

[0398] or

[0399] , wherein the immune checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

[0402] The composition according to any one of paragraphs

[0334] to

[0344] ,

[0360] to

[0369] , and

[0396] to

[0401] , further comprising an immunomodulator or an adjuvant.

[0403] The composition according to paragraph

[0402] , wherein the immunomodulator is a costimulatory ligand, TNF ligand, Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB.

[0404] The composition according to paragraph

[0402] , wherein the immunomodulator is at least one cancer cell or cancer cell extract.

[0405] The composition according to paragraph

[0404] , wherein the cancer cell is autologous to the subject in need of the composition.

[0406] The composition according to paragraph

[0405] , wherein the cancer cell is lysed or exposed to UV irradiation.

[0407] The composition according to paragraph

[0402] , further comprising an adjuvant.

[0408] wherein the adjuvant is: poly(I:C), polyICLC, STING agonist, 1018ISS, aluminum salt, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosome and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, an acrylic or methacrylic polymer, a copolymer of maleic anhydride, and QS21 stimulin, the composition according to paragraph

[0407] .

[0409] wherein the adjuvant, when administered to a subject, induces humoral, the composition according to paragraph

[0407] or

[0408] .

[0410] wherein the adjuvant, when administered to a subject, induces helper T cell type 1, the composition according to paragraph

[0409] .

[0411] A method of inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 1 or Table 2, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell according to any of paragraphs

[0291] to

[0410] .

[0412] A method for inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 3 or Table 4, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell described in any of paragraphs

[0291] to

[0410] .

[0413] A method for inhibiting the growth of tumor cells expressing a tumor-specific epitope defined in Table 5 or Table 6, the method comprising contacting the tumor cells with a peptide, polynucleotide, delivery system, vector, composition, antibody, or cell described in any of paragraphs

[0291] to

[0410] .

[0414] A method for treating cancer or treating cancer in a subject in need of initiation, enhancement, or prolongation of an anti-tumor response, or initiating, enhancing, or prolonging an anti-tumor response, the method comprising administering to the subject a peptide, polynucleotide, vector, composition, antibody, or cell described in any of paragraphs

[0291] to

[0410] .

[0415] The method according to any of paragraphs

[0411] to

[0414] , wherein the subject is human.

[0416] The method according to paragraph

[0415] , wherein the subject has cancer.

[0417] The method according to paragraph

[0416] , wherein the cancer is selected from the group consisting of genitourinary cancer, kidney cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or osteosarcoma, hematological neoplasm, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), smoldering myeloma (SMM), breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, and small cell lung cancer.

[0418] The method according to any one of paragraphs

[0411] to

[0417] , wherein the subject has undergone surgical removal of a tumor.

[0419] The method according to any one of paragraphs

[0411] to

[0418] , wherein the peptide, polynucleotide, vector, composition, or cell is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration.

[0420] The method according to paragraph

[0419] , wherein the peptide, polynucleotide, vector, composition, or cell is administered to an anatomical site that drains into a lymph node basin.

[0421] The method according to paragraph

[0420] , wherein the administration is made to a plurality of lymph node basins.

[0422] The method according to any one of paragraphs

[0411] to

[0421] , wherein the administration is by a subcutaneous or intradermal route.

[0423] The method according to paragraph

[0419] , wherein a peptide is administered.

[0424] The method according to paragraph

[0423] , wherein the administration is intratumoral.

[0425] The method according to paragraph

[0419] , wherein a polynucleotide and, optionally, RNA are administered.

[0426] The method according to paragraph

[0419] or

[0425] , wherein the polynucleotide is administered intravenously.

[0427] The method according to paragraph

[0419] , wherein the cell is a T cell or a dendritic cell.

[0428] The method according to paragraph

[0419] or

[0427] , wherein the peptide or polynucleotide comprises an antigen-presenting cell targeting moiety.

[0429] The method according to any one of paragraphs

[0411] to

[0428] , further comprising administering to the subject at least one immune checkpoint inhibitor.

[0430] The method according to paragraph

[0429] , wherein the checkpoint inhibitor is a biological therapeutic agent or a small molecule.

[0431] The method according to paragraph

[0429] or

[0430] , wherein the checkpoint inhibitor is selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody, and a fusion protein, or a combination thereof.

[0432] The method according to any one of paragraphs

[0429] to

[0431] , wherein the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands, or a combination thereof.

[0433] The method according to any one of paragraphs

[0429] to

[0432] , wherein the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands, or a combination thereof.

[0434] The method according to any one of paragraphs

[0429] to

[0433] , wherein two or more checkpoint inhibitors are administered.

[0435] The method according to paragraph

[0434] , wherein the checkpoint inhibitor is (i) ipilimumab or tremelimumab, and (ii) nivolumab.

[0436] The method according to any one of paragraphs

[0429] to

[0435] , wherein the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order.

[0437] The method according to paragraph

[0436] , wherein the peptide, polynucleotide, vector, composition, or cell is administered before the checkpoint inhibitor.

[0438] The method according to paragraph

[0436] , wherein the peptide, polynucleotide, vector, composition, or cell is administered after the checkpoint inhibitor.

[0439] The method according to paragraph

[0436] , wherein the administration of the checkpoint inhibitor continues throughout the antigen peptide, polynucleotide, vector, composition, or cell therapy.

[0440] The method according to any one of paragraphs

[0429] to

[0439] , wherein the antigen peptide, polynucleotide, vector, composition, or cell therapy is administered to a subject that partially responds or does not respond to checkpoint inhibitor therapy.

[0441] The method according to any one of paragraphs

[0411] to

[0428] , wherein the composition is administered intravenously or subcutaneously.

[0442] The method according to any one of paragraphs

[0429] to

[0440] , wherein the checkpoint inhibitor is administered intravenously or subcutaneously.

[0443] The method according to any one of paragraphs

[0429] to

[0441] , wherein the checkpoint inhibitor is administered subcutaneously within about 2 cm of the administration site of the composition.

[0444] The method according to paragraph

[0443] , wherein the composition is administered to the same draining regional lymph node as the checkpoint inhibitor.

[0445] The method according to any one of paragraphs

[0411] to

[0444] , further comprising administering an additional therapeutic agent to the subject before, simultaneously with, or after treatment with the peptide, polynucleotide, vector, composition, or cell.

[0446] The method according to paragraph

[0445] , wherein the additional agent is a chemotherapeutic agent, an immunomodulatory agent, an immunometabolic modifier, a targeted therapy, radiation, an anti-angiogenic agent, or an agent that reduces immunosuppression.

[0447] The method according to paragraph

[0446] , wherein the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an antimetabolite, or an antimitotic agent.

[0448] The method according to paragraph

[0445] , wherein the additional agent is an anti-glucocorticoid-induced tumor necrosis factor family receptor (GITR) agonist antibody or antibody fragment, ibrutinib, docetaxel, cisplatin, or cyclophosphamide.

[0449] The method according to any one of paragraphs

[0411] to

[0448] , which induces a CD4+ T cell immune response.

[0450] The method according to any one of paragraphs

[0411] to

[0449] , which induces a CD4+ T cell immune response and a CD8+ T cell immune response.

[0451] A method for stimulating an immune response in a subject, comprising administering an effective amount of the modified cell or composition according to any one of paragraphs

[0394] to

[0410] .

[0452] The method according to paragraph

[0451] , wherein the immune response is a cytotoxic and / or humoral immune response.

[0453] The method according to paragraph

[0451] , which stimulates a T cell-mediated immune response in a subject.

[0454] The method according to paragraph

[0453] , wherein the T cell-mediated immune response is directed against target cells.

[0455] The method according to paragraph

[0454] , wherein the target cells are tumor cells.

[0456] The method according to any one of paragraphs

[0451] to

[0455] , wherein the modified cells are transfected or transduced in vivo.

[0457] The method according to any one of paragraphs

[0451] to

[0456] , wherein the modified cells are transfected or transduced ex vivo.

[0458] The method according to any one of paragraphs

[0451] to

[0457] , wherein the modified cells are autologous patient T cells.

[0459] The method according to paragraph

[0458] , wherein the autologous patient T cells are obtained from a patient who has received an antigen peptide or nucleic acid vaccine.

[0460] The method according to paragraph

[0459] , wherein the antigen peptide or nucleic acid vaccine comprises at least one individualized antigen.

[0461] The method according to paragraph

[0460] , wherein the antigen peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 1 or Table 2.

[0462] The method according to paragraph

[0460] , wherein the antigen peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 3 or Table 4.

[0463] The method according to paragraph

[0460] , wherein the antigen peptide or nucleic acid vaccine comprises at least one additional antigenic peptide listed in Table 5 or Table 6.

[0464] The method according to paragraph

[0461] , wherein the at least one additional antigenic peptide listed in Table 1 or Table 2 is a retroviral antigenic peptide.

[0465] The method according to paragraph

[0462] , wherein the at least one additional antigenic peptide listed in Table 3 or Table 4 is a non-mutated overexpressed antigenic peptide.

[0466] The method according to paragraph

[0463] , wherein the at least one additional antigenic peptide listed in Table 5 or Table 6 is a viral antigenic peptide.

[0467] The method according to any one of paragraphs

[0461] to

[0466] , wherein the patient has received a chemotherapeutic agent, an immunomodulatory agent, an immunometabolic modifier, a targeted therapy, or radiation before and / or during receiving the antigen peptide or nucleic acid vaccine.

[0468] The method according to any one of paragraphs

[0459] to

[0467] , wherein the patient receives a treatment using at least one checkpoint inhibitor.

[0469] The method according to any one of paragraphs

[0459] to

[0468] , wherein the autologous T cells are obtained from a patient who has already received at least one round of T cell therapy containing an antigen.

[0470] The method according to any one of paragraphs

[0459] to

[0469] , further comprising adoptive T cell therapy.

[0471] The method according to paragraph

[0470] , wherein the adoptive T cell therapy comprises autologous T cells.

[0472] The method according to paragraph

[0471] , wherein the autologous T cells are targeted to tumor antigens.

[0473] The method according to paragraph

[0470] or

[0471] , wherein the adoptive T cell therapy further comprises allogeneic T cells.

[0474] The method according to paragraph

[0473] , wherein the allogeneic T cells are targeted against tumor antigens.

[0475] The adoptive T cell therapy according to any one of paragraphs

[0470] to

[0474] , wherein the adoptive T cell therapy is administered before the checkpoint inhibitor.

[0476] A method for evaluating efficacy according to any one of paragraphs

[0411] to

[0475] , comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administering the modified cells; (ii) measuring the number or concentration of target cells in a second sample obtained from the subject after administering the modified cells; and (iii) determining an increase or decrease in the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample.

[0477] The method according to paragraph

[0476] , wherein treatment efficacy is determined by monitoring clinical outcome; increased, enhanced or prolonged anti-tumor activity by T cells; increased number of anti-tumor T cells or activated T cells compared to the number before treatment; B cell activity; CD4 T cell activity; or a combination thereof.

[0478] The method according to paragraph

[0477] , wherein treatment efficacy is determined by monitoring biomarkers.

[0479] The method according to paragraph

[0478] , wherein the biomarker is selected from the group consisting of CEA, Her-2 / neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA125, CA19.9, CA15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, and B-type natriuretic peptide.

[0480] The method according to paragraph

[0477] , wherein the clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor enlargement, recurrence or spread; or a combination thereof.

[0481] The method according to paragraph

[0477] , wherein the treatment effect is predicted by the presence of T cells, or by the presence of a gene signature indicative of T cell inflammation, or a combination thereof.

[0482] A method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof, the method comprising administering to the subject:

[0483] A peptide, polynucleotide, vector, composition, antibody, or cell according to any of paragraphs

[0291] to

[0410] ; and

[0484] At least one checkpoint inhibitor.

[0485] The method according to paragraph

[0482] , further comprising administering an immunomodulatory factor or an adjuvant.

[0486] The method according to paragraph

[0483] , wherein the immunomodulatory factor or adjuvant is selected from the group consisting of poly(I:C), polyICLC, STING agonist, 1018ISS, aluminum salt, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymer, copolymer of maleic anhydride, and QS21 Stimulon, costimulatory ligand, TNF ligand, Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB.

[0487] The method according to paragraph

[0484] , wherein the immunomodulatory factor or adjuvant is polyICLC.

[0488] The method according to any one of paragraphs

[0482] to

[0485] , wherein the checkpoint inhibitor is an anti-PDl antibody or antibody fragment.

[0489] The method according to paragraph

[0486] , wherein the inhibitor of the PD-1 pathway is nivolumab.

[0490] The method according to any one of paragraphs

[0482] to

[0485] , wherein the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment.

[0491] The method according to paragraph

[0488] , wherein the anti-CTLA4 antibody is ipilimumab or tremelimumab.

[0492] The method according to any one of paragraphs

[0482] to

[0489] , comprising administering both an anti-PD1 antibody and an anti-CTLA4 antibody.

[0493] The method according to any one of paragraphs

[0482] to

[0489] , wherein the administration of the checkpoint inhibitor is started before the start of the administration of the peptide, polynucleotide, vector, composition, antibody, or cell.

[0494] The method according to any one of paragraphs

[0482] to

[0489] , wherein the administration of the checkpoint inhibitor is started after the start of the administration of the peptide, polynucleotide, vector, composition, antibody, or cell.

[0495] The method according to any one of paragraphs

[0482] to

[0489] , wherein the administration of the checkpoint inhibitor is started simultaneously with the start of the administration of the peptide, polynucleotide, vector, composition, antibody, or cell.

[0496] The method according to any one of paragraphs

[0482] to

[0493] , wherein the peptide, polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously.

[0497] The method according to any one of paragraphs

[0482] to

[0493] , wherein the checkpoint inhibitor is administered intravenously or subcutaneously.

[0498] The method according to any one of paragraphs

[0482] to

[0495] , wherein the checkpoint inhibitor is administered subcutaneously within about 2 cm of the administration site of the peptide, polynucleotide, vector, composition, antibody, or cell.

[0499] The method according to paragraph

[0496] , wherein the peptide, polynucleotide, vector, composition, antibody, or cell is administered to the same draining regional lymph node as the checkpoint inhibitor.

[0500] A kit comprising an antigen therapeutic agent according to any one of paragraphs

[0291] to

[0410] .

[0501] The method according to paragraph

[0414] , wherein the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, lung squamous cell carcinoma, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer.

[0502] The method according to paragraph

[0414] , wherein the cancer is selected from the group consisting of melanoma, lung squamous cell carcinoma, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC.

[0503] The method according to paragraph

[0414] , wherein the cancer is selected from the group consisting of cervical cancer, head and neck cancer, anal cancer, gastric cancer, Burkitt lymphoma, and nasopharyngeal cancer.

[0504] This specification provides an immunogenic vaccine composition comprising a peptide comprising at least 8 contiguous amino acids of an array in any one of Tables 1-6. In some embodiments, the peptide is a synthetic peptide. In some embodiments, the peptide is a recombinant peptide. In some embodiments, the peptide comprises a sequence derived from an endogenous retroviral protein. In some embodiments, the peptide comprises a sequence derived from an exogenous viral protein. In some embodiments, the peptide comprises a sequence of a protein expressed by cancer cells of a subject having cancer, wherein the protein is expressed by cancer cells at a higher level than the level expressed by non-cancer cells of the subject. In some embodiments, the peptide is 100 amino acids in length or less. In some embodiments, the peptide is about 5 to about 50 amino acids in length or about 15 to about 35 amino acids in length. In some embodiments, the peptide is about 30 amino acids in length or less or about 15 amino acids in length or less. In some embodiments, the peptide comprises a sequence that binds to major histocompatibility complex (MHC) class I with a binding affinity of less than about 500 nM. In some embodiments, the peptide comprises a sequence that binds to major histocompatibility complex (MHC) class II with a binding affinity of less than about 1000 nM. In some embodiments, the peptide further comprises a non-natural amino acid adjacent to at least 8 contiguous amino acids. In some embodiments, the composition further comprises a second peptide comprising at least 8 contiguous amino acids of an array in any one of Tables 1-6, wherein the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM. In some embodiments, the peptides are linked using a polyglycine or polyserine linker. In some embodiments, the second antigenic peptide binds to MHC class I or class II with a binding affinity of less than about 1000 nM or less than about 500 nM. In some embodiments, the peptide further comprises a modification that increases in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.In some embodiments, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polyallylation, HESylation, recombinant PEG mimetic, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of an amino acid mimic, or addition of a non-natural amino acid. In some embodiments, the peptide comprises a modification that increases targeting by antigen-presenting cells. In some embodiments, the antigen-presenting cell is a dendritic cell. In some embodiments, the modification that increases targeting by dendritic cells is DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, or CD1a marker. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 30 peptides, each comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. In some embodiments, the composition comprises 2 to 20 peptides, each comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6.In some embodiments, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, 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, or at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 additional antigenic peptides. In some embodiments, the additional antigenic peptides are specific for the tumor of an individual patient. In some embodiments, the additional antigenic peptides are selected by identifying sequence differences between the genome, exome, and / or transcriptome of a patient's tumor sample and the genome, exome, and / or transcriptome of a non-tumor sample. In some embodiments, identifying the sequence differences comprises performing next-generation sequencing. Provided herein is a composition comprising an antigen-presenting cell comprising a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. In some embodiments, the antigen-presenting cell is a dendritic cell.

[0505] Provided herein is an in vivo delivery system comprising the composition described herein. In some embodiments, the delivery system comprises a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, or liposomes. In some embodiments, the cell-penetrating peptide is a TAT peptide, herpes simplex virus VP22, transportan, or Antp.

[0506] The present specification provides an immunogenic vaccine composition comprising a recombinant polynucleotide encoding a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. In some embodiments, the recombinant polynucleotide is RNA, optionally self-amplifying RNA. In some embodiments, the RNA is modified to increase stability, increase cell targeting, increase translation efficiency, adjuvant activity, cytosolic accessibility, and / or decrease cytotoxicity. In some embodiments, the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC content, incorporation of modified nucleosides, incorporation of a 5'-cap or cap analog, and / or incorporation of an unmasked polyA sequence.

[0507] The present specification provides a composition comprising a cell comprising a recombinant polynucleotide encoding a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6.

[0508] The present specification provides a composition comprising a vector comprising a polynucleotide comprising a sequence encoding a peptide comprising at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the polynucleotide is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion. In some embodiments, the virus is an adeno-associated virus, herpes virus, lentivirus, or pseudotype thereof.

[0509] The present specification provides an in vivo delivery system comprising the composition described herein. In some embodiments, the delivery system comprises spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.

[0510] As used herein, a T cell receptor (TCR) that specifically binds to a peptide:MHC complex, wherein the peptide of the peptide of the peptide:MHC complex comprises at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. A T cell receptor (TCR) is provided.

[0511] As used herein, a T cell comprising a T cell receptor (TCR) that specifically binds to a peptide:MHC complex, wherein the peptide of the peptide of the peptide:MHC complex comprises at least 8 contiguous amino acids of a sequence in any one of Tables 1-6. In some embodiments, the T cell is a helper T cell or a cytotoxic T cell. In some embodiments, the T cell is an autologous patient T cell.

[0512] A method of treating cancer in a subject in need of cancer treatment, the method comprising administering to the subject a composition described herein, wherein the subject has cancer cells expressing a protein comprising at least 8 contiguous amino acids of an array in any one of Tables 1-6, is provided. In some embodiments, the subject is human. In some embodiments, the cancer is selected from the group consisting of genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, triple-negative breast cancer (TNBC), malignant melanoma, Merkel cell carcinoma, or osteosarcoma, hematological neoplasm, multiple myeloma, smoldering myeloma (SMM), acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, and small cell lung cancer. In some embodiments, the method further comprises administering to the subject at least one immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

Claims

[Claim 1] The invention described in the specification.

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