Common neoantigen

Abstract

To provide a method that can overcome some of the drawbacks of vaccines containing common tumor antigens.SOLUTION: A pharmaceutical composition comprising multiple neoantigen peptides and a pharmaceutically acceptable carrier, the neoantigen peptides each comprising a tumor-specific neoepitope capable of binding to an HLA protein of interest, each tumor-specific neoepitope comprising a tumor-specific mutation present in a tumor, and a) the composition comprises a neoantigen peptide comprising a tumor-specific mutation present in at least 1% of subjects in a population of subjects with cancer, (b) the composition comprises a neoantigen peptide comprising a tumor-specific neoepitope that binds to an HLA protein present in at least 5% of subjects in the population, and (c) the composition comprises at least one neoantigen peptide capable of eliciting an immune response against a tumor present in at least 5% of subjects in a population of subjects with cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications and references This application claims priority and benefits of U.S. Provisional Application No. 62 / 179,877, filed on 20 May 2015, and U.S. Provisional Application No. 62 / 389,377, filed on 23 February 2016.

[0002] The aforementioned application, and all cited documents in the application or its examination procedures ("Application Citations"), and all documents cited or referenced in the Application Citations, and all documents cited or referenced herein ("Specified Citations"), and all documents cited or referenced in the Specified Citations, together with any manufacturer's instructions, descriptions, product specifications, and product sheets relating to any product mentioned herein or in any document referred herein, are incorporated herein by reference and may be used in practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as each individual document is specifically and individually indicated as to be incorporated by reference.

[0003] The present invention relates to methods and compositions for treating neoplasms (e.g., tumors) using, in particular, at least one neoantigen peptide suitable for treating a substantial proportion of subjects in a population affected by cancer. [Background technology]

[0004] Approximately 1.6 million Americans are diagnosed with neoplasms each year, and in 2013, it was estimated that around 580,000 people in the U.S. would die from this disease. In recent decades, the detection, diagnosis, and treatment of neoplasms have improved dramatically, leading to a significant increase in survival rates for many types of neoplasms. However, only about 60% of those diagnosed with neoplasms are still alive five years after starting treatment, making neoplasms the second leading cause of death in the United States.

[0005] Currently, there are numerous existing cancer therapies, including ablation techniques (e.g., surgical procedures, cryo / thermal treatment, ultrasound, radiofrequency, and radiation) and chemical techniques (e.g., pharmaceuticals, cytotoxic / chemotherapeutic agents, monoclonal antibodies, and various combinations thereof). Unfortunately, these treatments often carry serious risks, toxic side effects, and extremely high costs, in addition to their uncertain effectiveness. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Cancer therapies that attempt to target cancer cells using the patient's own immune system (e.g., cancer vaccines) are attracting increasing attention because such therapies can mitigate / eliminate some of the drawbacks described herein. Cancer vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., cytokines or TLR ligands) that work together to induce antigen-specific cytotoxic T cells, which then target and destroy tumor cells. Current cancer vaccines may contain common tumor antigens, which are native proteins (i.e., proteins encoded by the DNA of all normal cells in an individual) that are selectively expressed or overexpressed in tumors found in many individuals. While such common tumor antigens are useful for identifying specific types of tumors, they are not ideal as immunogens for targeting T cell responses against specific tumor types because they contribute to an immune-weakening autotolerance effect. Vaccines containing tumor-specific and patient-specific neoantigens address some of the drawbacks of vaccines containing common tumor antigens. This can be resolved. However, using patient-specific neoantigens requires sequencing of the genome of each individual subject and the manufacture of personalized compositions containing the combination of neoantigens present in these individual subjects. Therefore, there is still a need for improvements in methods and compositions for delivering cancer vaccines.

[0007] Any reference or specification of any document in this application does not constitute an admission that such document is available as prior art for the present invention. [Means for solving the problem]

[0008] Preferred descriptions (features) and embodiments of the present invention are shown below in this specification. Each description and embodiment of the present invention as defined herein may be combined with any other description and / or embodiment unless expressly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or description indicated as preferred or advantageous. In this specification, the present invention is specifically incorporated by any one or any combination of one or more of the descriptions and / or embodiments below and any other descriptions and / or embodiments.

[0009] The object of the present invention is to provide methods and compositions for treating a population of cancer patients by inducing an immune response that targets cancer. In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one neoantigen peptide and a pharmaceutically acceptable carrier, wherein each of the at least one neoantigen peptide comprises a tumor-specific neoepitope capable of binding to a target HLA protein, and each tumor-specific neoepitope comprises a tumor-specific mutation present in the tumor. The composition may contain one neoantigen peptide. In other embodiments, the composition may contain more than 100 neoantigen peptides. Preferably, the composition contains about 20 neoantigen peptides. This at least one neoantigen peptide may contain a tumor-specific mutation. This mutation may be repetitive. Preferably, this mutation is present in the majority of the population. The repetitive mutation may be based on a mutation present in tumors in at least 1% of subjects in a population of subjects affected by cancer. The composition may contain at least one neoantigen peptide comprising a tumor-specific neoepitope that binds to an HLA protein present in at least 5% of subjects in a population of subjects affected by cancer. In addition, the composition may contain at least one neoantigen peptide capable of inducing an immune response against tumors present in at least 5% of subjects in a population of subjects affected by cancer. The ability to induce an immune response refers to the ability of the immune system to present the antigen to lymphocytes. For the immune system to present the antigen, this antigen must be presented by the subject's HLA protein. For the composition to induce an immune response against a tumor, the tumor must contain a mutation in which the antigen is expressed. For the composition to benefit a population that needs it, this population must include subjects that express an HLA allele capable of binding to at least one neoantigen peptide present in the composition, and this population must include subjects that have tumors with mutations in which a neoantigen epitope exists in the neoantigen peptide.

[0010] This composition may be specific to a population of subjects suffering from cancer that shares certain characteristics. This population may have cancer or have a specific cancer. This population may share a common set of HLA subtypes. This population may share HLA subtypes based on ethnicity. Although not bound by theory, the proportion of HLA types in a population can be predicted based on ethnicity without testing. Although not bound by theory, different populations express different HLA types that can bind to different neoantigen peptides. Therefore, a composition may be formulated to benefit the majority of one population, but this composition may not benefit another population. While not restrictive, different cancers contain different mutations, and therefore, using a composition tailored to a specific cancer can yield greater benefits to a population with one type of cancer compared to a population containing multiple types. In one embodiment, this population includes adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and kidney renal papillary cell carcinoma. Carcinoma (KIRP), acute myeloid leukemia (LAML), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM), ovarian serous cystadenocarcinoma (ovarian) The patient has serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), uterine corpus endometrioid carcinoma (UCEC), or uterine carcinosarcoma (UCS).

[0011] In one embodiment, the target population has CLL, and at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary diseases of "CLL," and at least one of a set of six of the at least one tumor-specific mutations can be found in 17.49% of the subjects in the CLL population. The target population may also have BLCA, and at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary diseases of "BLCA," and at least one of a set of six of the at least one tumor-specific mutations can be found in 26.92% of the subjects in this population. The target population may also have BRCA, and at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary diseases of "BRCA," and at least one of a set of 18 of the at least one tumor-specific mutations can be found in 36.04% of the subjects in this population. The target population may be affected by COAD, and at least one tumor-specific mutation may be found in 27.14% of subjects in this population, with at least one mutation from a set of three of the mutations listed in Table 8 and the exemplary disease of "COAD". The target population may be affected by GBM, and at least one tumor-specific mutation may be found in 34.36% of subjects in this population, with at least one mutation from a set of fourteen of the mutations listed in Table 8 and the exemplary disease of "GBM". The target population may be affected by HNSC, and at least one tumor-specific mutation may be found in 21.61% of subjects in this population, with at least one mutation from a set of ten of the mutations listed in Table 8 and the exemplary disease of "HNSC".The target population may be affected by KIRC, and at least one tumor-specific mutation may be found in 6% of the subjects in this population, with at least one of a set of four of the mutations listed in Table 8 and any combination of the exemplary disease “KIRC”. The target population may also be affected by LAML. Furthermore, at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary disease "LAML", and at least one of the 11 sets of at least one tumor-specific mutation can be found in 47.45% of subjects in this population. The subject population may be affected by LUAD, and at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary disease "LUAD", and at least one of the 11 sets of at least one tumor-specific mutation can be found in 33.42% of subjects in this population. The subject population may be affected by LUSC, and at least one tumor-specific mutation includes any combination of the mutations in Table 8 and the exemplary disease "LUSC", and at least one of the 2 sets of at least one tumor-specific mutation can be found in 7.87% of subjects in this population. The target population may be affected by OV, and at least one tumor-specific mutation may be found in 22.78% of subjects in this population, with at least one mutation from a set of 10 of the mutations listed in Table 8 and the exemplary disease "OV". The target population may be affected by READ, and at least one tumor-specific mutation may be found in 20.51% of subjects in this population, with at least one mutation from a set of 2 of the mutations listed in Table 8 and the exemplary disease "READ". The target population may be affected by SKCM, and at least one tumor-specific mutation may be found in 90.91% of subjects in this population, with at least one mutation from a set of 64 of the mutations listed in Table 8 and the exemplary disease "SKCM". The target population may have UCEC, and at least one tumor-specific mutation may be found in 67.74% of subjects in this population, with at least one of 30 tumor-specific mutations being found in any combination of the mutations in Table 8 and the exemplary diseases of “UCEC”.The target population may be affected by ACC, and at least one tumor-specific mutation may be found in 50% of the subjects in this population, with at least one mutation from a set of 161 of the mutations listed in Table 8 and the exemplary diseases of "ACC". The target population may be affected by CESC, and at least one tumor-specific mutation may be found in 23.71% of the subjects in this population, with at least one mutation from a set of 4 of the mutations listed in Table 8 and the exemplary diseases of "CESC". The target population may be affected by CRC, and at least one tumor-specific mutation may be found in 56.65% of the subjects in this population, with at least one mutation from a set of 15 of the mutations listed in Table 8 and the exemplary diseases of "CRC". The target population may be affected by DLBCL, and at least one tumor-specific mutation may be found in 13.79% of subjects in this population, with at least one mutation from a set of two from the mutations listed in Table 8 and the exemplary disease of "DLBCL". The target population may be affected by KICH, and at least one tumor-specific mutation may be found in 50% of subjects in this population, with at least one mutation from a set of 24 from the mutations listed in Table 8 and the exemplary disease of "KICH". The target population may be affected by KIRP, and at least one tumor-specific mutation may be found in 42.24% of subjects in this population, with at least one mutation from a set of nine from the mutations listed in Table 8 and the exemplary disease of "KIRP".The target population may have LIHC, and at least one tumor-specific mutation includes any combination of mutations in Table 8 and the exemplary disease “LIHC”, with at least one of a set of two of the at least one tumor-specific mutation present in 6.57% of the subjects in this population. It can be found. The target population may have MM, and at least one tumor-specific mutation can be found in 23.9% of subjects in this population, with at least one tumor-specific mutation including any combination of mutations in Table 8 and the exemplary disease of "MM". At least one of the six sets of at least one tumor-specific mutation can be found in 39.85% of subjects in this population. The target population may have PRAD, and at least one tumor-specific mutation can be found in 39.85% of subjects in this population, with at least one tumor-specific mutation including any combination of mutations in Table 8 and the exemplary disease of "PRAD". At least one of the 150 sets of at least one tumor-specific mutation can be found in 48.79% of subjects in this population. The target population may be affected by TGCT, and at least one tumor-specific mutation may be found in 51.61% of subjects in this population, with at least one mutation from a set of 14 tumor-specific mutations, including any combination of mutations from Table 8 and the exemplary disease of "TGCT". The target population may be affected by THCA, and at least one tumor-specific mutation may be found in 69.88% of subjects in this population, including any combination of mutations from Table 8 and the exemplary disease of "THCA". The target population may be affected by UCS, and at least one tumor-specific mutation may be found in 16.07% of subjects in this population, including any combination of mutations from Table 8 and the exemplary disease of "UCS".The population may have PAAD, and at least one tumor-specific mutation may be found in 50% of the subjects in this population, with at least one tumor-specific mutation being any combination of the mutations in Table 8 and the exemplary diseases of "PAAD".

[0012] In another embodiment, at least one tumor-specific mutation may occur in at least 500 patients per year in a cancer-affected population, and at least one mutation may be one of the mutations listed in Table 9 for this population. At least one neoantigen peptide may be one of the peptides listed in Table 9.

[0013] In another embodiment, the cancer-affected population is treated with a drug or therapy. This cancer-affected population may have already been treated, is currently being treated, or is selected to be treated with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, or anti-estrogen therapy.

[0014] In another embodiment, the composition comprises at least one neoantigen peptide capable of inducing an immune response against tumors present in at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of subjects in a population of subjects suffering from cancer.

[0015] In another embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of subjects in this population have at least one tumor-specific mutation present in the composition, and at least Each of the following components contains 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of an HLA protein that binds to tumor-specific neoepitopes present in the composition.

[0016] In one embodiment, tumor-specific mutations include splice-variant mutations, point mutations, and / or frameshift mutations. In another embodiment, tumor-specific mutations include drug resistance mutations. In one embodiment, the neoantigen peptide includes not only the resulting mutated neoantigen protein sequence, but also the long peptide region surrounding and containing the mutation, and further includes all adjacent segments within the long peptide (see Tables 1-4). In one embodiment, tumor-specific mutations are present in one or more genes encoding proteins selected from the group consisting of: programmed death ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEK1, MEK2, NRAS, RAC1, and ESR1. In one embodiment, tumor-specific mutations are present in one or more genes listed in any of the tables presented herein. In one embodiment, at least one tumor-specific mutation is derived from alternative splicing of PD-L1 or AR. In one embodiment, at least one tumor-specific mutation is derived from splice variants sPD-L1, AR-V1, or AR-V7.In one embodiment, at least one tumor-specific mutation is a drug resistance mutation selected from the group consisting of: BTK / C481S, EGFR / T790M, BCR-Abl / T315I, BCR-Abl / Y253H, BCR-Abl / E255K, BCR-Abl / E255V, c-kit / T670I, PIK3CA / E545K, PIK3CA / E542K, HER2 / G776(YVMA), HER2 / E545K, EML4-ALK / G1269A, KRAS / G12V / D, ALK / L1196M, ALK / G1202R, ALK / S1206Y, ALK / 1151T(in s),ALK / F1174C,ROS1 / G2032R,AKT1 / E17K,BRAF / V600E,MEK1 / Q56P,MEK1 / E203K ,MEK1 / C121S,MEK1 / V60E,MEK1 / G128V,MEK1 / V154I,MEK1 / P124S,MEK1 / P124L,NR AS / Q61K / L / R,NRAS / T58I,MEK2 / C125S,RAC1 / P29S,ESR1 / S463P,AR / V534E,AR / P5 35H, AR / L536Q, AR / L536R, AR / Y537C, AR / Y537S, AR / Y537N, AR / D538G and AR / F876L. In one embodiment, drug resistance mutations are induced by treatment with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, or anti-estrogen therapy. In another embodiment, the subject has drug resistance mutations prior to treatment.

[0017] In another embodiment, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 neoantigen peptides. The composition may contain 15 to 20 neoantigen peptides. The composition may contain more than 100, 200, or 300 neoantigen peptides. Each neoantigen peptide may be about 5 to about 50 amino acids long.

[0018] In another embodiment, the pharmaceutical composition is an immunogenic or vaccine composition. The pharmaceutical composition may further comprise an immunomodulator or adjuvant. This immunomodulator or adjuvant may be polyICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, cyclic dinucleotide, e.g., STING, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, J uvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide The following may be selected: ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, and Aquila's QS21 stimulon.

[0019] In another embodiment, the pharmaceutical composition comprises one or more neoantigen peptides as defined in Tables 1, 2, 3, or 4.

[0020] In one embodiment, each tumor-specific neoepitope has a K content of less than 500 nM. D It binds to HLA-A, -B, or -C, or HLADRB, HLADBM, or XXXXX.

[0021] In another aspect, the present invention relates to a method for treating or preventing a tumor of a subject requiring such treatment, which involves administering any of the pharmaceutical compositions described herein to the subject.

[0022] In one embodiment, a method is provided for treating or preventing a tumor in a patient in need thereof, comprising administering to the patient a composition comprising at least one neoantigen peptide and a pharmaceutically acceptable carrier, wherein each of the at least one neoantigen peptide comprises a tumor-specific neoepitope capable of binding to a target HLA protein, each tumor-specific neoepitope comprising a tumor-specific mutation present in the tumor, the composition comprising at least one neoantigen peptide comprising a tumor-specific mutation present in tumors of at least 1% of subjects in a population of subjects with cancer, the composition comprising at least one neoantigen peptide comprising a tumor-specific neoepitope capable of binding to an HLA protein present in at least 5% of subjects in a population of subjects with cancer, and the composition comprising at least one neoantigen peptide capable of inducing an immune response against tumors present in at least 5% of subjects in a population of subjects with cancer.

[0023] In one embodiment, the target population includes adrenocortical carcinoma (ACC), urothelial carcinoma of the bladder (BLCA), invasive breast cancer (BRCA), squamous cell carcinoma of the cervix and cervical adenocarcinoma (CESC), adenocarcinoma of the colon (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), squamous cell carcinoma of the head and neck (HNSC), chromophobe cells of the kidney (KICH), clear cell carcinoma of the kidney (KIRC), Renal papillary cell carcinoma (KIRP) and acute myeloid leukemia (LAML) of the kidney; hepatocellular carcinoma (LIHC) of the liver; lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM); serous cystadenocarcinoma (OV) of the ovary; pancreatic adenocarcinoma (PAAD); prostate adenocarcinoma (PRAD); rectal adenocarcinoma (READ); cutaneous melanoma (SKCM) of the skin; gastric adenocarcinoma (STAD); germ cell tumor of the testis (TGCT); thyroid adenocarcinoma (THCA); and endometrial carcinoma (uterine corpus cancer). The subjects have endometrioid carcinoma (UCEC) or uterine carcinosarcoma (UCS). In one embodiment, the subjects have a solid tumor, which may be clear cell renal carcinoma (ccRCC), melanoma, sarcoma, or cancer of the bladder, colon, brain, breast, head and neck, endometrium, lung, ovary, pancreas, or prostate. In one embodiment, the subjects have a humoral tumor, which may be non-Hodgkin lymphoma or leukemia.

[0024] In one embodiment, a group of cancer patients has been, is being, or is selected to be treated with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, or anti-estrogen therapy. .

[0025] In one embodiment, at least one neoantigen peptide can induce an immune response against tumors present in at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of subjects in a population of subjects with cancer. In one embodiment, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of subjects in this population have at least one tumor-specific mutation present in the composition, and at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of subjects in this population have at least one HLA protein that binds to a tumor-specific neoepitope present in the composition.

[0026] In another embodiment, tumor-specific mutations include splice-variant mutations, point mutations, and / or frameshift mutations. These tumor-specific mutations may be drug-resistance mutations. These tumor-specific mutations may be present in one or more genes encoding proteins selected from the group consisting of: programmed death ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEK1, MEK2, NRAS, RAC1, and ESR1. These tumor-specific mutations may be present in one or more genes listed in any of the tables. These at least one tumor-specific mutation may originate from alternative splicing of PD-L1 or AR. These at least one tumor-specific mutation may originate from splice variants sPD-L1, AR-V1, or AR-V7.

[0027] In one embodiment, at least one tumor-specific mutation is a drug resistance mutation selected from the group consisting of: BTK / C481S, EGFR / T790M, BCR-Abl / T315I, BCR-Abl / Y253H, BCR-Abl / E255K, BCR-Abl / E255V, c-kit / T670I, PIK3CA / E545K, PIK3CA / E542K, HER2 / G776(YVMA), HER2 / E545K, EML4-ALK / G1269A, KRAS / G12V / D, ALK / L1196M, ALK / G1202R, ALK / S1206Y, ALK / 1151T(ins ), ALK / F1174C, ROS1 / G2032R, AKT1 / E17K, BRAF / V600E, MEK1 / Q56P, MEK1 / E203K, MEK1 / C121S,MEK1 / V60E,MEK1 / G128V,MEK1 / V154I,MEK1 / P124S,MEK1 / P124L,NRA S / Q61K / L / R, NRAS / T58I, MEK2 / C125S, RAC1 / P29S, ESR1 / S463P, AR / V534E, AR / P5 35H, AR / L536Q, AR / L536R, AR / Y537C, AR / Y537S, AR / Y537N, AR / D538G and AR / F876L. These drug-resistance mutations can be induced by treatment with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, or anti-estrogen therapy.

[0028] In another embodiment, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 neoantigen peptides. In a preferred embodiment, the composition comprises 15 to 20 neoantigen peptides.

[0029] In another embodiment, each neoantigen peptide is approximately 5 to 50 amino acids long.

[0030] In another embodiment, the composition is an immunogenic or vaccine composition. For example, this The immunogenic or vaccine composition may contain an immunomodulator or adjuvant. This immunomodulator or adjuvant may be selected from the group consisting of: poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, cyclic dinucleotides, e.g., STING, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vectors, PLGA microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, and Aquila's QS21 stimulon.

[0031] In one embodiment, the composition comprises one or more neoantigen peptides as defined in Tables 1, 2, 3, or 4.

[0032] In one embodiment, each tumor-specific neoepitope has a K content of less than 500 nM. D It binds to HLA-A, -B, or -C, or HLADRB, HLADBM, or XXXXX.

[0033] In another embodiment, the present invention provides a method for prophylactic cancer treatment, comprising selecting an anticancer agent for a patient in need thereof, selecting the agent from the group consisting of ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, and anti-estrogen therapy, and prophylactically administering to a subject a pharmaceutical composition comprising a neoantigen peptide derived from a drug resistance mutation associated with the selected anticancer agent, before the drug resistance mutation can be detected.

[0034] A common neoantigen immunogenic composition can be administered by subcompositions, each containing a portion of the neoantigen, and these subcompositions can be administered to different locations in the subject or patient; for example, a composition containing 20 different neoantigens can be administered by four subcompositions, each containing five of the 20 different neoantigens, and these four subcompositions can be administered to attempt to deliver each subcomposition to the individual aspiration lymph nodes of the patient, e.g., each of the upper and lower limbs (e.g., the thigh or upper thigh or near the buttocks or lumbar region on each side of the patient's body), thereby limiting competition between neoantigens. Naturally, the number of locations and, consequently, the number of subcompositions can vary, and for example, a person skilled in the art may consider an administration having a fifth administration site in or near the spleen, and a person skilled in the art may change the locations so that only one, two or three (e.g., each upper limb and one lower limb, each lower limb and one upper limb, each lower limb and no upper limbs, or both upper limbs only) are used. The common neoantigen immunogenic compositions administered at the various intervals described above may be different formulations, and the subcompositions administered to different locations in the subject or patient during a single dose may be different compositions. For example, the initial dose may be the entire common neoantigen immunogenic composition, and subsequent doses may be vectors (e.g., viral vectors or plasmids) that express one or more antigens in vivo. Similarly, when administering different subcompositions to different locations in the patient or subject, some subcompositions may contain all antigens, and some subcompositions may contain vectors (e.g., viral vectors or plasmids) that express one or more antigens in vivo. Furthermore, some compositions and subcompositions may contain both vectors (e.g., viral vectors or plasmids) that express one or more antigens in vivo and all antigens. Some vectors (e.g., poxviruses) that express one or more antigens in vivo may have an immunostimulatory or adjuvant effect, and such vectors may The contained compositions or subcompositions may be autoadjuvants. Furthermore, by altering the nature of how antigens are presented to the immune system through administration, the immune system can be "primed" and then "boosted." In this document, when the term "vaccine" is used, the present invention is intended to encompass immunogenic compositions, and when the term "patient or subject" is used, such individual is intended to be a patient or subject who requires the treatments, administrations, compositions, and generally the present invention disclosed herein.

[0035] Furthermore, the present invention applies to the use of any type of expression vector, for example, viral expression vectors, for example, poxviruses (e.g., orthopoxvirus or avipoxvirus, for example, vaccinia virus, for example, modified vaccinia ankara or MVA, MVA-BN, NYVAC as described in International Publication A-92 / 15672, fowlpox, for example, trovax, canarypox, for example, ALVAC (International Publication A-95 / 27780 and International Publication A-92 / 15672), pigeonpox, swinepox, etc.), adenoviruses, AAVs, herpesviruses, and lentiviruses; or plasmid or DNA or nucleic acid molecular vectors. Some cytoplasmic vectors, such as poxvirus vectors, may be advantageous. However, adenoviruses, AAVs, and lentiviruses may also be advantageous for use in the implementation of the present invention.

[0036] In ready-to-use, particularly reconstituted, common neoantigen immunogenic compositions, vectors, such as viral vectors, are present in amounts within the scope of those skilled in the art from the present disclosure and the knowledge in the art (including that found in the patents and scientific literature cited herein).

[0037] The whole antigen or vector, such as a recombinant live vaccine, may exist in a freeze-dried form that allows for its storage and is reconstituted immediately before use in a solvent or excipient that may contain an adjuvant as discussed herein.

[0038] Accordingly, the subject matter of the present invention is also a vaccination or immunization set or kit comprising a separately packaged freeze-dried vaccine and a reconstitution solution for the freeze-dried vaccine, which advantageously comprises an adjuvant compound as discussed herein.

[0039] The subject matter of the present invention is also a vaccination or immunization method comprising, or essentially comprising, a step of administering the vaccine or immunogenic composition according to the present invention at one or more administration rates, for example, by parenteral routes, preferably subcutaneous, intramuscular, or intradermal routes, or by mucosal routes. Optionally, the method includes a preliminary step of reconstituting a freeze-dried common neoantigen immunogenic composition (e.g., lyophilized whole antigen or vector) into a solution, which also advantageously includes an adjuvant.

[0040] In one embodiment, a common neoantigen immunogenic composition is administered at a dose of approximately 10 μg to 1 mg per 70 kg individual with respect to each neoantigen peptide. In another embodiment, the common neoantigen immunogenic composition is administered at an average weekly dose level of approximately 10 μg to 2000 μg per 70 kg individual with respect to each neoantigen peptide. In another related embodiment, this administration is intravenous. In one embodiment, the common neoantigen immunogenic composition is administered intravenously or subcutaneously.

[0041] In another embodiment, the method further includes (a) obtaining a sample of tumor tissue from each subject, (b) detecting one or more tumor-specific mutations in the sample, and (c) selecting a subject from the population of subjects for treatment with at least one neoantigen peptide if at least one tumor-specific mutation is detected in the sample derived from the subject.

[0042] In another embodiment, the method further includes (a) detecting the HLA allotype present in each subject, and (b) selecting a subject from a population of subjects for treatment with at least one neoantigen peptide if one or more HLA allotypes present in the subject bind to one or more tumor-specific neoepitopes present in at least one neoantigen peptide.

[0043] Embodiments of the present invention relate to compositions and methods using a common neoantigen, which, unlike common innate (non-mutagenic) antigens derived from genes differentially expressed in tumors, possess desired properties (e.g., not susceptible to central tolerance-reducing effects and high tumor specificity). This is because the neoantigen is expressed only in tumor tissue, for example, because it is generated by tumor-specific mutations or splicing defects. Such splice variants or mutations can generate immunogenic epitopes across various HLA alleles, thus covering a significant proportion of the population. Furthermore, since these mutations may be present in a significant proportion of subjects affected by cancer, the compositions described herein do not require whole-genome sequencing of the subject and can be used as "off-the-shelf" products for treating multiple subjects. For example, the method may simply involve detecting one or more of the specific mutations present in the composition from a tumor sample derived from the subject, and administering the composition to a subject in which at least one mutation is present. This is in contrast to methods that use patient-specific neoantigen mixtures, which require sequencing of the entire genome or exome of each subject and the manufacture of individualized treatment compositions.

[0044] Other embodiments relate to combination therapy using a method of treatment with the common neoantigen composition of the present invention in conjunction with a current drug regimen. This common neoantigen composition can be administered prophylactically. In one embodiment, a patient in need is treated with chemotherapy and / or targeted therapy in combination with the common neoantigen immunogenic composition before drug resistance mutations can be detected. This common neoantigen immunogenic composition may be formulated to contain neoantigen peptides specific to resistance mutations associated with the selected therapy. In another embodiment, this common neoantigen composition is administered before treating a target with chemotherapy and / or targeted therapy to induce an immune response against cells with drug resistance mutations before such cells develop. This administration may be sequential or sequential, or substantially simultaneous or substantially simultaneous. For example, the administration of the common neoantigen immunogenic composition and the administration of cancer treatment may be substantially simultaneous or substantially simultaneous. Alternatively, the administration of the common neoantigen immunogenic composition can be on a one-time schedule, for example, weekly, bi-weekly, every three weeks, monthly, every other month, every quarter (every three months), every third (every four months), every five months, twice a year (every six months), every seven months, every eight months, every nine months, every ten months, every eleven months, every year, etc. The administration of cancer treatment can be on a separate schedule, typical of treatments where the subject or patient has two different treatment schedules progressing simultaneously, and the administration of the common neoantigen immunogenic necrosis composition and the administration of cancer treatment can be sequential or continuous. In a preferred embodiment, the subject may be treated with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK, or anti-estrogen therapy.

[0045] In another embodiment, the present invention provides a diagnostic method for early detection and tracking of cancer progression by determining the presence of at least one neoantigen peptide of the present invention in a patient sample. The patient sample may be derived from blood, sputum, saliva, urine, tumor tissue, lymph, semen, or feces.

[0046] In one embodiment, the diagnostic method is used before administering the common neoantigen composition described herein. The diagnostic method may include comparing the amount of common neoantigen mutations in a series of at least two samples taken during cancer treatment and / or treatment with the common neoantigen composition. While not bound by theory, the effectiveness of the treatment can be determined using an increase or decrease in common neoantigen mutations.

[0047] In one embodiment, mutant genes can be detected using PCR-based methods or sequencing. Mutations in transcribed neoantigen genes can be detected using reverse transcription PCR (RT-PCR). In addition, the presence of mutations can be determined using any sequencing technique. In a preferred embodiment, pyrosequencing is used. The present invention also provides a kit comprising primers that are specific to sequences containing neoantigen mutations.

[0048] In another embodiment, this mutation is detected by an immunological detection method. This mutation can be detected using an antibody specific to the common neoantigen mutation. This antibody can be conjugated to an array. This array can contain antibodies for detecting two or more of the common neoantigen mutations of the present invention. This antibody can be configured for use in an ELISA assay. Therefore, a composition or kit containing an antibody that specifically recognizes the common neoantigen of the present invention can be provided.

[0049] In another aspect, the present invention provides a method for treating or preventing tumors in a target population in need thereof, comprising administering to the target a drug comprising an extracellular ligand-binding domain that recognizes a tumor-specific neoepitope having an incidence of at least 1% of the target population, including a tumor-specific mutation. The drug may be an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a CAR, or a T cell receptor. The antibody or antibody fragment may be humanized, fully humanized, or a chimeric. The antibody fragment may be a nanobody, Fab, Fab', (Fab')2, Fv, ScFv, a bispecific antibody, a triplicate antibody, a quadruplicate antibody, Bis-scFv, a minibody, a Fab2, or a Fab3 fragment. The tumor-specific mutation may be one of the mutations listed in Table 9 for any population. The tumor-specific mutation may be intracellular in a gene comprising an extracellular domain. This tumor-specific mutation can be FGFR3 S249C, ERBB3 V104M, EGFR L858R, MUC4 H4205Q, PDGFRA R483fs, TMEM52 23_26LLPL>L, or PODXL 28_30PSP>P. This tumor-specific mutation can be located within the extracellular domain. This tumor-specific mutation includes FGFR3 S249C or ERBB3 V104M. Although not constrained by theory, the presence of a neoepitope in a protein with an extracellular domain allows this neoepitope to exist on the cell surface. Although not constrained by theory, the presence of a neoepitope within the extracellular domain allows this neoepitope to exist on the cell surface.

[0050] The present invention is further described by the following numbered paragraphs.

[0051] 1. Isolated neoantigen peptides containing tumor-specific neoepitopes as defined in Tables 1-9, which are not native polypeptides.

[0052] 2. Isolated neoantigen peptides with a length of 100 amino acids or less, containing tumor-specific neoepitopes as defined in Tables 1-9.

[0053] 3. An isolated neoantigen peptide as described in paragraph 1 or 2, having a length of approximately 5 to 50 amino acids.

[0054] 4. An isolated neoantigen peptide, approximately 15 to 35 amino acids in length, as described in any one of paragraphs 1 to 3.

[0055] 5. An isolated neoantigen peptide described in paragraph 4, with a length of approximately 15 amino acids or less.

[0056] 6. The isolated neoantigen peptide described in paragraph 5, having a length of approximately 8 to 11 amino acids.

[0057] 7.9 or 10 amino acid length, isolated neoantigen peptide as described in paragraph 6.

[0058] 8. An isolated neoantigen peptide described in any one of paragraphs 1-7 that binds to major histocompatibility complex (MHC) class I.

[0059] 9. The isolated neoantigen peptide described in paragraph 8, which binds to MHC class I with a binding affinity of less than approximately 500 nM.

[0060] 10. An isolated neoantigen peptide, approximately 30 amino acids or less in length, as described in any one of paragraphs 1-3.

[0061] 11. The isolated neoantigen peptide described in paragraph 10, having a length of approximately 6 to 25 amino acids.

[0062] 12. The isolated neoantigen peptide described in paragraph 11, having a length of approximately 15 to 24 amino acids.

[0063] 13. The isolated neoantigen peptide described in paragraph 11, having a length of approximately 9 to 15 amino acids.

[0064] 14. An isolated neoantigen peptide that binds to MHC class II, as described in any one of paragraphs 1-3 and 10-13.

[0065] 15. The isolated neoantigen peptide described in paragraph 14, which binds to MHC class II with a binding affinity of less than approximately 1000 nM.

[0066] 16. An isolated neoantigen peptide described in any one of paragraphs 1 to 15, further comprising flanking amino acids.

[0067] 17. The above flanking amino acids are not naturally occurring flanking amino acids, but isolated neoantigen peptides as described in paragraph 16.

[0068] 18. An isolated neoantigen peptide described in any one of paragraphs 1 to 17, which is linked to at least a second neoantigen peptide.

[0069] 19. The isolated neoantigen peptide described in paragraph 18, wherein the peptide is linked using a polyglycine or polyserine linker.

[0070] 20. The isolated neoantigen peptide described in paragraph 18 or 19, wherein the second neoantigen peptide described above binds to MHC class I or class II with a binding affinity of less than 1000 nM.

[0071] 21. The isolated neoantigen peptide described in paragraph 20, wherein the second neoantigen peptide described above binds to MHC class I or class II with an affinity of less than approximately 500 nM.

[0072] 22. Both neoepitopes are human leukocyte antigens (HLA)-A, -B, -C, -DP, -An isolated neoantigen peptide as described in paragraph 20 or 21, which binds to -DQ or -DR.

[0073] 23. The isolated neoantigen peptide described in any one of paragraphs 20 to 22, wherein the isolated neoantigen peptide and the second neoantigen peptide bind to class I HLA, or the isolated neoantigen peptide and the second neoantigen peptide bind to class II HLA.

[0074] 24. The isolated neoantigen peptide described in any one of paragraphs 20 to 22, wherein the isolated neoantigen peptide binds to class II HLA and the second neoantigen peptide binds to class I HLA, or the isolated neoantigen peptide binds to class I HLA and the second neoantigen peptide binds to class II HLA.

[0075] 25. An isolated neoantigen peptide as described in any one of paragraphs 1 to 24, further comprising modifications that increase in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.

[0076] 26. The isolated neoantigen peptide described in paragraph 25, wherein the above modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylated HESation, recombinant PEG mimetic, Fc fusion, albumin fusion, attachment of nanoparticles, encapsulation of nanoparticles, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, oxidation of side chains, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimetic, or addition of non-natural amino acids.

[0077] 27. The target cells are antigen-presenting cells, as described in paragraph 25, and the isolated neoantigen peptide.

[0078] 28. The antigen-presenting cells are dendritic cells, and the isolated neoantigen peptide is as described in paragraph 27.

[0079] 29. The isolated neoantigen peptide described in paragraph 29, which targets the dendritic cells using the CD141, DEC205, or XCR1 marker.

[0080] 30. A pharmaceutical composition comprising at least one neoantigen peptide and a pharmaceutically acceptable carrier, wherein each of the at least one neoantigen peptides comprises a tumor-specific neoepitope capable of binding to a target HLA protein, and each of the tumor-specific neoepitopes comprises a tumor-specific mutation present in the tumor. (a) The composition comprises at least one neoantigen peptide containing a tumor-specific mutation present in tumors of at least 1% of the subjects in a population of subjects suffering from cancer, (b) The composition comprises at least one neoantigen peptide containing a tumor-specific neoepitope that binds to an HLA protein present in at least 5% of the subjects in a population of subjects affected by cancer, or (c) The above composition comprises at least one neoantigen peptide capable of inducing an immune response against tumors present in at least 5% of subjects in a population of subjects suffering from cancer. Pharmaceutical composition.

[0081] 31. The above target population includes adrenocortical carcinoma (ACC), urothelial carcinoma of the bladder (BLCA), invasive breast cancer (BRCA), squamous cell carcinoma and cervical adenocarcinoma (CESC), adenocarcinoma of the colon (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), chromophobe cell carcinoma of the kidney (KICH), clear cell carcinoma of the kidney (KIRC), papillary cell carcinoma of the kidney (KIRP), acute myeloid leukemia (LAML), hepatocellular carcinoma of the liver (LIHC), and lung adenocarcinoma. The pharmaceutical composition described in paragraph 30, for patients suffering from cancer (LUAD), squamous cell carcinoma of the lung (LUSC), multiple myeloma (MM), serous cystadenocarcinoma of the ovary (OV), adenocarcinoma of the pancreas (PAAD), adenocarcinoma of the prostate (PRAD), adenocarcinoma of the rectum (READ), cutaneous melanoma of the skin (SKCM), adenocarcinoma of the stomach (STAD), germ cell tumor of the testis (TGCT), adenocarcinoma of the thyroid gland (THCA), endometrial carcinoma of the uterine body (UCEC), or carcinosarcoma of the uterus (UCS).

[0082] 32. The pharmaceutical composition described in paragraph 30 or 31, in which the population suffering from the above-mentioned cancer has been, is being, or is selected to be treated with an anticancer drug, at the discretion of ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK inhibitor, or anti-estrogen therapy.

[0083] 33. The pharmaceutical composition according to any one of paragraphs 30 to 33, wherein the tumor-specific mutations include splice variant mutations, point mutations, and / or frameshift mutations.

[0084] 34. The pharmaceutical composition according to any one of paragraphs 30 to 33, wherein the at least one neoantigen peptide comprises at least one neoantigen peptide derived from a long peptide region adjacent to and containing the tumor-specific mutation, and includes all adjacent segments within the long peptide.

[0085] 35. The above tumor-specific mutation is present in one or more genes listed in Tables 1-9, and is a pharmaceutical composition as described in any one of paragraphs 30-34.

[0086] 36. The pharmaceutical composition according to any one of paragraphs 30 to 35, wherein the composition comprises at least one neoantigen peptide as defined in any of Tables 1 to 9.

[0087] 37. The pharmaceutical composition according to any one of paragraphs 30 to 36, wherein the tumor-specific mutation is present in one or more genes encoding a protein selected from the group consisting of programmed death ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEK1, MEK2, NRAS, RAC1, and ESR1.

[0088] 38. The pharmaceutical composition according to paragraph 37, wherein at least one tumor-specific mutation is derived from alternative splicing of PD-L1 or AR.

[0089] 39. The pharmaceutical composition according to paragraph 38, wherein at least one tumor-specific mutation is derived from the splice variant sPD-L1, AR-V1, or AR-V7.

[0090] 40. The pharmaceutical composition according to any one of paragraphs 30 to 39, wherein the tumor-specific mutations include drug resistance mutations.

[0091] 41. At least one tumor-specific mutation is present in BTK / C481S, EGFR / T790M, BCR-Abl / T315I, BCR-Abl / Y253H, BCR-Abl / E255K, BCR-Abl / E255V, c-kit / T670I, PIK3CA / E545K, PIK3CA / E542K, HER2 / G776(YVMA), HER2 / E 545K,EML4-ALK / G1269A,KRAS / G12V / D,ALK / L1196M,ALK / G1202R,ALK / S1206Y,ALK / 1151T(ins) ,ALK / F1174C,ROS1 / G2032R,AKT1 / E17K,BRAF / V600E,MEK1 / Q56P,MEK1 / E203K,MEK1 / C121S,MEK The pharmaceutical composition described in paragraph 40, wherein the drug resistance mutation is selected from the group consisting of 1 / V60E, MEK1 / G128V, MEK1 / V154I, MEK1 / P124S, MEK1 / P124L, NRAS / Q61K / L / R, NRAS / T58I, MEK2 / C125S, RAC1 / P29S, ESR1 / S463P, AR / V534E, AR / P535H, AR / L536Q, AR / L536R, AR / Y537C, AR / Y537S, AR / Y537N, AR / D538G, and AR / F876L.

[0092] 42. The pharmaceutical composition according to any one of paragraphs 30 to 41, wherein the at least one tumor-specific mutation has an incidence rate of at least 500 patients per year in a target population affected by cancer, and the at least one mutation includes the mutations listed in Table 9 with respect to the population.

[0093] 43. The pharmaceutical composition according to paragraph 42, wherein the above-mentioned neoantigen peptide comprises at least one peptide listed in Table 9.

[0094] 44. (a) The above group of subjects is suffering from CLL, (b) Structural structures of SF3B1:p.K700E,MYD88:p.L273P,NOTCH1:p.P 2514fs,ABCA11P:p.E901D,CHILDREN:p.D3823E,ZNF814:p.E348D,CHILDREN:p.V 1220I,AHNAK:p.H1203N,ANKRD30A:p.A232V,APOOL:p.I138L,EGR2:p.H397N,MKI67:p.H2213D,NRAS:p.Q61R,PLIN4:p.M691V,XPO1:p.E571K,ZCRB1:1. p.L76F,ZNF700:p.N652H,ZNF700:p.Q654R,ZNF844:p.D458H,AHNAK:p.A4046V,ANKRD36:p.P337R,C1orf170:p.T203I,CAST:p.D639E,EGR2:p.E369 K,GPR123:p.L630P,IKZF3:p.L162R,MUC4:p.P4224R,OR9Q1:p.M34L,PKD2:p.Y486F,PRAMEF11:p.R104Q,SYNJ1:p.I681F,TP53:p.R248Q,TP53:p.R24 8W,TRPV2:p.L627del,ZNF254:p.S498A,ZNF732:p.A459T,ZNF749:p.E530Q,ZNF845:p.M423I,ABCA11P:p.G900E,ACRC:p.E243D,ACRC:p.A244V,ACS L3:p.T188S,ADAMTS2:p.D948N,AGAP6:p.S127I,AHNAK:p.A2114G,ANKRD36:p.D1014Y,ARID3A:p.G550fs,ARID4A:p.D1154E,ATP2B4:p.R183H,ATRNL 1:p.L1244F,BNC1:p.Y937N,BRAF:p.K601N,BTLA:p.Q86K,C14orf177:p.G90V,C2orf44:p.N456K,C3orf15:p.R552Q,CACNA2D1:p.Y376N,CALD1:p.E3 40K,CCDC15:p.P488H,CCDC79:p.N440T,CCNB3:p.A932T,CD109:p.L470Q,CD209:p.Q189L,CKAP2:p.*684K,CMA1:p.I81K,CMIP:p.A230T,CNTNAP4:p.I12F,CRYM:p.*315K,DICER1:p.E1705K,DPCR1:p.L716P,EIF3A:p.M1093L,EIF4G3:p.R8H,ETFDH:p.I281F,EWSR1:p.Y656C,F5:p.L1332P,F5:p.L1253F,FAM50A:p.H317R,FBXL13:p.S102R,FBXW7:p.R465H,FHL1:p.D184E,FILIP1:p.I522K,FRG1B:p.Q39K,GNB1:p.I80T,GPR110:p.R443G,GPR98:p.Y6152F,HDGFL1:p.188_189insA,IGF2BP2:p.T186S,IL1R2:p.L364fs,KIAA1109:p.L4680P,KRAS:p.G13D,KRTAP19-1:p.G61S,MAF:p.G5. 3fs,MAGEC1:p.L609H,MAP2K1:p.K57N,MED12:p.L36R,MED12:p.G44S,METAP2:p.Y137N,METTL9:p.Y57F,MGP:p.V15L,MKI67:p.R2222K,MUC16: p.T11005I,MUC4:p.S3941N,MUC4:p.S3941G,MUC4:p.V3091L,MUC4:p.S2951Y,MUC4:p.A2841S,MUC4:p.S2760A,MUC4:p.T2335M,MUC4:p.T1627K ,MUC4:p.T1547S,MUC4:p.H1133Q,MYD88:p.M240T,NEDD4L:p.P194del,NEFH:p.S704T,NRG4:p.G21fs,OR2A25:p.S105C,OR4C16:p.Y63F,OR4N4: p.L150fs,PABPC1:p.K254fs,PIWIL1:p.P372fs,PLCD3:p.E499fs,PLEKHB1:p.S146P,PPIL4:p.S382R,PRDM4:p*802K,PRG4:p.N675H,PRKAB1:p .P104H,R3HDM2:p.S592G,R3HDM2:p.S588N,R3HDM2:p.R206W,RPS2:p.R200G,RPTN:p.G364S,SF3B1:p.K666E,SF3B1:p.N626Y,SF3B1:p.Y623C, SIX3:p.I27L,SLC39A7:p.L456fs,SLC6A9:p.R94K,TFG:p.A382V,TGOLN2:p.K83R,TGOLN2:p.T80S,TLR2:p.D327V,TNKS2:p.T619fs,TP53:p.R27 3H,TP53:p.C242F,TP53:p.R175H,TWISTNB:p.H306Q,UBXN7:p.A276V,WDR78:p.N110K,XIRP2:p.V3008E,ZNF382:p.H186Q,ZNF578:p.R306H,ZNF 578:p.G311S,ZNF578:p.H334R,ZNF700:p.S649C,ZNF705A:p.D298N,ZN F836:p.K608Q, and ZNF836:p.I571N This is a great way to get the best results A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0095] 45. (a) The above group of subjects is infected with BLCA, (b) At least one of the above tumor-specific mutations is PIK3CA:p.E545K, FGFR3:p.S249C, TP53:p.R248Q, PIK3CA:p.E542K, RXRA:p.S427F, ZNF814:p.D404E, FBXW7:p.R505G, NOTCH2:p.P6fs, TP53:p.E285K, ANKRD30A:p.A353P, C3orf70:p.S6L, EFCAB6:p.R379K, ERCC2:p.N238S, FAM47C:p. Q225E,FOXQ1:p.S135L,HLA-A:p.Q78R,MUC4:p.H4205Q,OTUD4:p.T909I,SLAMF1:p.S277fs,SPRED3:p.S128del,TMCO2:p.S15fs,TP 53:p.R280T,TP53:p.E271K,TP53:p.A159V,ZNF706:p.I8N,ZNF706:p.R3P,ACACB:p.E2318Q,ACPP:p.E321K,ACRC:p.A264V,ADAMTS 2:p.23_24insL,AFF3:p.E919K,AHNAK:p.S4150F,AHNAK:p.D2889H,AHNAK:p.V1940A,ALX4:p.R126Q,ANKRD12:p.E627K,ANKRD32:p .T999N,ARID1A:p.S614L,ASXL2:p.117_118SS>S,ATP12A:p.R858C,ATP9A:p.R519Q,BCAS3:p.T214M,BPI:p.M255I,CACNG8:p.V146 G,CAMSAP1:p.T466fs,CDC27:p.I91fs,CDKN1A:p.E44fs,CEP192:p.S2058L,CGB8:p.T18A,CHRNA3:p.L23del,CHST4:p.D352N,CLIP 1:p.S1018fs,COX6A1:p.S8L,CREBBP:p.D1435H,CRIPAK:p.M48fs,CSPG5:p.D119N,CUL1:p.E485K,DLC1:p.S741T,DLL3:p.D318H,D OPEY2:p.E1196K,ECM1:p.E266K,EEF1A2:p.Y418S,EEF2K:p.E673K,EMILIN1:p.R27G,ERBB2:p.S310F,ERBB3:p.M91I,ERBB3:p.V104L,ERBB3:p.D2 97Y,ERCC2:p.Y14C,FAM155A:p.Q86del,FAM43B:p.E272del,FASTKD3:p.Q625E,FBXW7:p.S546L,FGFR3:p.R248C,FGFR3:p.G380R,FGFRL1:p.H479f s,GBE1:p.M587I,GIMAP1-GIMAP5:p.S311C,GNA13:p.R200G,H1FOO:p.A214fs,HEATR7B2:p.E1109K,HIST1H1D:p.I81M,HRAS:p.G12D,HRCT1:p.H92 P,ILF3:p.E484K,KCNK2:p.S6W,KIAA0907:p.Q446P,KIF23:p.E350K,KLF5:p.S118L,KLHL15:p.D185G,LAMA4:p.E639K,LILRA1:p.H410Y,LILRB1:p .L479del,LLGL2:p.P955fs,LPIN1:p.S974L,LRRC16A:p.D227N,LRTM2:p.S139L,LURAP1L:p.55_56insGGG,MAGEC1:p.P553del,MCL1:p.E171del,M N1:p.S472L,MUC7:p.A191V,MVP:p.E412K,NBPF10:p.E3455K,NFE2L2:p.E79K,NFE2L2:p.R34G,NOS1AP:p.Q306del,OR2T35:p.V319fs,OR4N2:p.L1 50fs,PABPC3:p.K333fs,PAX3:p.S197L,PBX2:p.E70K,PBXIP1:p.H729del,PCDP1:p.E537K,PEX1:p.I370fs,PHLDA3:p.E82K,PLEKHM2:p.S459L,PLVAP:p.A321V,POLR3B:p.L372F,POTEC:p.R477Q,PPL:p.H326Y,PPP1R15A:p.E196K,PRDM16:p.E271Q,PRIC285:p.E1289Q,PRMT8:p.S31P,PUF60:p.S396L,RAB11FIP4:p.S596L,RAD51C:p.D167N,RAD51C:p.Y224H,RALGPS1:p.R381Q,RARS2:p.R6C,RBM26:p.P644A,RERE:p. K176N,RXRA:p.S427Y,SERPINA12:p.R211G,SF3B1:p.E902K,SLC6A9:p.R243W,SLC9A5:p.L447F,SPESP1:p.F121L,SRPRB:p .G14S,SYN2:p.A34del,SYTL2:p.I440M,TAB3:p.R211T,TAF1B:p.R292C,TAOK2:p.L981del,TAS1R3:p.E525K,TAS2R9:p.E1 63Q,TBC1D1:p.S71F,TBC1D2B:p.R920Q,TFPI2:p.R222C,TM6SF1:p.S15W,TMEM131:p.K640fs,TMEM19:p.G331fs,TP53:p.R2 73C,TP53:p.R248W,TP53:p.R175H,TP53:p.K132N,TRAM1:p.E41Q,TSKS:p.E513K,TTN:p.C20935G,UBOX5:p.S417L,UGP2:p .D262H,VGF:p.E433K,XAB2:p.E782K,XYLB:p.S87F,ZC3H4:p.E798K,ZNF208:p.K852E,ZNF208:p.I647S,ZNF626:p.G198E, This includes any combination of mutations selected from the group consisting of ZNF749:p.Q457E, ZNF761:p.H373R, ZNF799:p.T43A, ZNF799:p.W41G, ZNF799:p.E589G, ZNF844:p.P503R, ZNF845:p.M423T, ZNF845:p.T479M, ZNF860:p.H464R, ZNF878:p.S181R, ZNF91:p.R333H, and ZNF91:p.H305R. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0096] 46. ​​(a) The above group of subjects suffers from BRCA, (b) At least one of the above tumor-specific mutations is GATA3:p.L328fs, This includes any combination of frameshift mutations selected from the group consisting of GATA3:p.N334fs, GATA3:p.L344fs, GATA3:p.H400fs, GATA3:p.S408fs, GATA3:p.S430fs, GATA3:p.H434fs, GATA3:p.H435fs, and GATA3:p.S408fs. A pharmaceutical composition as described in any one of paragraphs 30 to 43.

[0097] 47.(a) The above group of subjects suffers from BRCA, (b)Schematic diagrams of 1 and PIK3CA:p.H1047R,PIK3CA:p.E545K,PIK3CA :p.E542K,AKT1:p.E17K,TP53:p.R175H,PIK3CA:p.N345K,PIK3CA:p.H104 7L,SF3B1:p.K700E,GATA3:p.S408fs,PIK3CA:p.E726K,TP53:p.Y220C,TP53:p.H193R,PIK3CA:p.Q546R,TP53:p.R273C,TP53:p.R248W,TP53:p.R27 3H,TP53:p.I195T,TP53:p.H179R,FGFR2:p.N549K,NUP93:p.E14K,PIK3CA:p.C420R,PIK3CA:p.E453K,PIK3CA:p.Q546K,TP53:p.V216M,TP53:p.C17 6F,CDH1:p.E243K,ERBB2:p.L755S,KRAS:p.G12V,PIK3CA:p.E545A,TBL1XR1:p.I141fs,TP53:p.G266E,TP53:p.R248Q,TP53:p.Y163C,TP53:p.C141 Y,TP53:p.G108fs,ACPP:p.R43W,AKT2:p.I289M,ARHGAP9:p.R137C,C9orf174:p.R136W,CDC42BPA:p.P675T,COL12A1:p.S395L,CRISPLD1:p.R222W, CT47B1:p.234_243EKLTEEATEE>E,CYP1A2:p.V483M,DAB2IP:p.E161K,DGKB:p.S13L,DMD:p.K1772N,DPEP1:p.V11L,ERBB2:p.S310F,ERBB2:p.D769Y ,ERBB3:p.E928G,ESYT1:p.R816W,FAM179A:p.A831T,FAM58BP:p.A70T,FMN2:p.S751F,GALNTL6:p.K567del,GATA3:p.L328fs,GATA3:p.N334fs,GAT A3:p.L344fs,GATA3:p.H400fs,GATA3:p.S408fs,GATA3:p.S430fs,GATA3:p.H434fs,GATA3:p.H435fs,GDAP1:p.T307A,GRB14:p.A300T,GUCY2C:p.G549C,IL17B:p.R34W,KCNB2:p.R231H,KIF1B:p.R1320W,KIF26B:p.V1113M,KLF4:p.K434Q,LY9:p.I69L,MAP2K4:p.S184L,MAP2K4:p.S251I,MAP2K4:p.T261fs,MAP3K1:p.L318fs,MAP3K1:p.I761fs,MAP3K1:p.V1346del,MAP3K1:p.L1384fs,MAPK13:p.E315K,MAPK4:p.V100M,MARCH5:p.R170C,MBP:p.E120K,MEFV:p.R377H,METTL15:p.Q53E,MS4A4A:p.V99M,MUC17:p.R4415H,MYH6:p.T847M,MYO5B:p.A405V,NARS2:p.P240R,NLGN4X:p.D382N,NLRC4:p.R288W,OR13G1:p.R258H,OR2AK2:p.V45I,OTOF:p.T388M,PACSIN2:p.Q331H,PALM2-AKAP2:p.A299T,PCDH19:p.R286C,PCDHGC5:p.D664N,PIK3CA:p.R88Q,PIK3CA:p.E110del,PIK3CA:p.K111del,PIK3CA:p.PVPHGLEDL447del,PIK3CA:p.L455fs,PIK3CA:p.M1004I,PIK3CA:p.M1043I,PIK3CA:p.N1044Y,PIK3R1:p.KPDL567del,PREX2:p.R363Q,PRRX1:p.A196V,PTEN:p.V317fs,RGSL1:p.V222I,RUNX1:p.R142fs,RUNX1:p. .D96fs,SCN2A:p.R36K,SLC25A32:p.Q83E,SLC25A45:p.G106C,STRA6:p.Q68R,STX6:p.H153D,TBX3:p.H187Y,TFPT:p.S252C,TINAG:p.R332W,TM EM71:p.R63Q,TP53:p.E286K,TP53:p.R282W,TP53:p.V272M,TP53:p.S241fs,TP53:p.C238fs,TP53:p.C238F,TP53:p.C238Y,TP53:p.Y234C,TP53 This includes any combination of mutations selected from the group consisting of :p.Y220S,TP53:p.R209fs,TP53:p.G199V,TP53:p.L194R,TP53:p.H193L,TP53:p.H193Y,TP53:p.V173L,TP53:p.V173M,TP53:p.K132N,TP53:p.R110fs,TUBD1:p.A200V,VLDLR:p.R231H,VWA3A:p.V955I,VWF:p.K1720N,XPO1:p.E571K, and ZNF268:p.F901del. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0098] 48. (a) The above group of subjects suffers from COAD, (b)Schematic diagrams of 1 and KRAS:p.G12D,BRAF:p.V600E,KRAS:p.G12V,A CVR2A:p.K435fs,GRB14:p.KKK295fs,SEC63:p.L532fs,TGFBR2:p.E125fs ,ATR:p.K771fs,ICA1:p.N204fs,KRAS:p.G12C,TP53:p.R175H,ABCA8:p.R842Q,ACTL7B:p.R354H,ACVR2A:p.K435fs,AIM2:p.K340fs,ALG2:p.S302Y, ANKIB1:p.K144fs,ARSG:p.V131I,ATP10D:p.R311H,AXIN2:p.W663fs,C5orf30:p.D4N,CACNG3:p.V134I,CASP5:p.K78fs,CC2D2A:p.R1284C,CDH10:p .E349K,DNMT1:p.E432K,DOCK2:p.G170R,DOCK5:p.E177K,EGR2:p.R390H, ERBB3:p.V104M,FAM135B:p.R884H,FBXW7:p.R505C,FBXW7:p.R465H,FHDC1 :p.R254W,FOXL1:p.N89K,HCN4:p.R525H,HLA-DMA:p.E84K,HTR3B:p.R236C,ITGA4:p.T673M,KIF18A:p.R17C,KIF20B:p.E991K,KLHL5:p.R326C,KRAS :p.A146T,KRAS:p.G13D,LPHN3:p.R1183Q,MAP2K4:p.R287H,MAPK8IP1:p.L217fs,MFSD5:p.R280Q,MUC16:p.R8606H,MYO6:p.D1180N,NAA25:p.S807Y ,NBPF14:p.V44L,NRAS:p.Q61K,NRAS:p.G13R,PAX3:p.T424M,PGAM1:p.R240H,PHF3:p.R1410I,PIK3CA:p.R88Q,PIK3CA:p.E545K,PIK3CA:p.H1047R, PLXNA3:p.V14fs,POSTN:p.R508C,PTPRU:p.D1434N,PYGO2:p.Q150fs,RBBP7:p.E274K,SFPQ:p.R611Q,SGSM1:p.F1117L,SLC25A40:p.R96Q,SLC8A1:p.This includes any combination of mutations selected from the group consisting of R431H, SLITRK3:p.S298L, SPATA22:p.S150L, ​​SUN3:p.E128K, TGFBR1:p.S241L, TP53:p.R273H, TP53:p.R273C, TP53:p.R248W, TRPV5:p.R492H, USP40:p.S851L, VPS13C:p.D1359Y, ZBTB24:p.L607I, ZNF434:p.R306C, ZNF443:p.R301I, ZNF484:p.R138C, and ZNF770:p.S441P. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0099] 49. (a) The above group of subjects is suffering from GBM, (b)Schematic diagrams of 1 and HSD17B7P2:p.N175S,IDH1:p.R132H,EGFR:p. A289V,EGFR:p.G598V,WASH3P:p.G175S,ZNF814:p.D404E,RPSA:p.Q111E, NBPF10:p.E3455K,TP53:p.R248Q,BRAF:p.V600E,EGFR:p.A289T,PRB2:p.N230del,RGPD5:p.P1760A,TP53:p.R175H,CHEK2:p.K373E,EGFR:p.R108K, EGFR:p.R222C,PIK3CA:p.E545K,PIK3R1:p.G376R,POTEC:p.K507E,SDHAP2:p.V195E,SLC6A10P:p.K88N,TP53:p.R282W,TP53:p.R273H,CD3EAP:p.K 219del,DST:p.R146C,EGFR:p.A289D,EGFR:p.H304Y,FRG1B:p.S71N,GOLGA8DP:p.A116E,KRTAP4-11:p.R121K,KRTAP4-11:p.S48R,MAP3K1:p.P324L, OGDH:p.I78fs,PODXL:p.S162fs,PSPH:p.V145I,SPINT1:p.A316V,TP53:p.R248W,TP53:p.G245S,TP53:p.Y220C,TP53:p.R158H,TSHZ2:p.A222T,UB C:p.L149R,ZDHHC4:p.R300H,ZNF844:p.R447P,AASS:p.T878fs,ABCC10:p.R570W,ADAM29:p.V205I,ADAMTS8:p.V524M,AGAP3:p.R766W,AICDA:p.Y14 4F,AK7:p.A159V,AK8:p.D243A,ANO2:p.R334C,AOX1:p.A507V,ARHGAP5:p.M691L,CALN1:p.V231I,CARM1:p.A202V,CD163L1:p.V721M,CD1D:p.L25f s,CD209:p.A283T,CDH18:p.A195T,CILP2:p.V553M,CIZ1:p.L89P,CLOCK:p.L123fs,COL6A5:p.T2224M,CSF2RB:p.G298S,CSMD3:p.E171K,CYP2D6:p.H352R,DCAF12L1:p.R335H,DCAF12L2:p.R246H,DPP10:p.V183I,DPY19L2P1:p.R378Q,DQX1:p.R505H,DRD5:p.S275R,DVL2:p.V66G,EFCAB6:p.R379K,EGFR:p.L62R,EGFR:p.R252C,EGFR:p.P596S,EGFR:p.P596L,EGFR:p.G598A,EGFR:p.E709K,EPHA1:p.A184T,ERC2:p.R20H,ESPNP:p.R627Q,FAM126B:p.R382H,FBN3:p.V886I,FGF14:p.T229M,FLG2:p.H1901fs,FLG:p.R2886H,FLNA:p.V1240M,FOXG1:p.H57del,FPR2:p.R54Q,FRG1B:p.K13N,FRG1B:p.A53T,GABRA6:p.V314I,GJB3:p.R160H,GLT8D2:p.A178V,GRM3:p.R183C,HERC1:p.R2330H,HNF1B:p.T417M,HTRA3:p.Q403R,IDH1:p.R132G,IFNA10:p.L80F,IFNA10:p.V79A,JHDM1D:p.R313H,JPH1:p.A395T,KEL:p.V411M,KIAA0907:p.R516fs,KIAA1704:p.D88del,KLK6:p.R120H,KRAS:p.G12D,KRTAP4-7:p.L121V,KRTAP4-7:p.L148V,KRTAP5-4:p.S131C,LAT2:p.L18W,LIMK2:p.R203H,LUM:p.R330C,MCOLN3:p.V141I,MGAT4B:p.T444P,MUC17:p.V77M,MUC17:p.3204_3205insP,MYO1D:p.T109M,MYO6:p.Q914fs,NAP1L5:p.140_141EE>E,NF1:p.F1658fs,NHP2L1:p.R84C,NLRP5:p.R737W,NPTX1:p.A263T,NUFIP2:p.Q29del,ODF4:p.R61C,OR11H12:p.H154P,OR2A7:p.V18I,OR2H1:p.V287I,OR2T12:p.R18. 4H,OR5D13:p.R236C,OR5P2:p.A100V,OR6N2:p.R293C,PASD1:p.A236del,PCDH11X:p.T486M,PCDHB13:p.P221L,PDGFRA:p.E229K,PDGFRB:p.S650L,PHC3:p.T35del,PIK3C2B:p.R287fs,PIK3CA:p.M1V,PIK3CA:p.R88Q,PIK3CA:p.M1043V,PIK3CA:p.H1047R,PIK3R1:p.K379N,PODNL1:p.A150V,POTEE:p.V166M,POTEG:p.R136H,PRKCD:p.G432fs,PROKR2:p.V297I,PTEN:p.C136Y,PTEN:p.S170N,PTEN:p.R173H,PTEN:p.T277I,PTEN:p.V317fs,PTPN14:p.E716del,R3HDM2:p.412_413QQ>Q,RAB11FIP5:p.R170H,RASAL3:p.R82H,RB1:p.N316fs,RDH8:p.A198V,REN:p.15_16LL>L,RIMBP2:p.R830H,SCAF11:p.E926fs,SCN7A:p.R1358H,SCNN1G:p.R564H,SDHAP2:p.R31C,SDHAP3:p.A66T,SEMG2:p.R292C,SH3RF2:p.R318C,SHB:p.A460T,SIGLEC10:p.T250M,SLC13A5:p.Q273P,SLC17A9:p.V324I,SLC22A9:p.R407Q,SLC26A3:p.V88I,SLC5A3:p.A302fs,SLC9A4:p.R631H,SPAM1:p.R346Q,SPEN:p.E803fs,SPTA1:p.A2011V,SUSD5:p.T513M,SYNE1:p.R8468H,TARSL2:p.G366D,TAS2R41:p.A255T,TAT:p.R367H,TFPI2:p.R206C,THSD7B:p.R90C,TMEM147:p.A92V,TMEM156:p.R81C,TMPRSS6:p.V302I,TNFSF9:p.A232T,TP53:p.C238F,TP53:p.C238Y,TP53:p.Y234C,TP53:p.V216M,TP53:p.H179R,TP53:p.This includes any combination of mutations selected from the group consisting of T155N, TRAPPPC10:p.K133fs, TTN:p.R21402W, TTN:p.V16403M, TUBBP5:p.V102M, TYRP1:p.T352fs, UBC:p.R73L, UGT2B28:p.P289H, USH2A:p.R3719H, WASH6P:p.L211V, ZFP42:p.V227I, ZFP42:p.T264M, ZNF181:p.V305G, ZNF280B:p.E400K, ZNF534:p.N583K, ZNF563:p.W208fs, ZNF844:p.F487L, and ZPBP:p.R154C. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0100] 50. (a) The above group of subjects is suffering from HNSC, (b) At least one of the above tumor-specific mutations is PIK3CA:p.E545K, PIK3CA:p.E542K, TP53:p.R175H, PIK3CA:p.H1047R, TP53:p.R282W, TP53:p.R248Q, TP53:p.R273H, TP53:p.R248W, TP53:p.G245S, RHOA:p.E40Q, EP300:p.D1399N, HRAS:p.G13V, MB21D2:p.Q311E, NFE2L2:p.E79Q, TP53:p.H179Y, FBXW7:p.R505G, HIST1H2BF:p.E77K, HRAS:p.G12D, MAPK1:p .E322K,NFE2L2:p.D29H,TP53:p.P278S,TP53:p.C242F,TP53:p.Y220C,TP53:p.H1 93L,TP53:p.H179R,TP53:p.V157F,TP53:p.R110L,AKNAD1:p.K620R,ANXA6:p.R231 Q,AP1G2:p.D243N,ATAD5:p.D441N,ATP6AP2:p.E119Q,B2M:p.M1I,BCL11A:p.E579K ,C1orf172:p.Y30fs,C7orf57:p.E30K,CCDC135:p.E313K,CDH12:p.P706T,CDH7:pQ 225K,CDK11B:p.E79del,CDKN2A:p.H83Y,CHCHD4:p.T79M,CIRH1A:p.S250I,CLSTN2:p.P759L,CRB1:p.L628fs,DENND5B:p.G1023E,DNAH5:p.Q1797E, DSP:p.R160G,EDA:p.L58F,EFCAB6:p.E1002K,ELF4:p.S415L,EP300:p.C1164Y,EPHA3:p.T802R,EPHA6:p.D952H,ERBB2:p.M916I,ESRRA:p.D219N,FA M101A:p.I89del,FBXO24:p.M553V,FCAR:p.V233M,GPANK1:p.Y351fs,GPR20:p.V300I,GPRASP1:p.S706L,GPRIN3:p.R633fs,GRID2:p.T649fs,GRM3:p.F682L,GUCY2F:p.S404L,HCRTR2:p.D100Y,HIST1H3C:p.K37M,HIST1H4C:p.R68P,HLX:p.S12T,HOXD10:p.Y151C,HPS3:p.K812N,HRAS:p.G12A,HRAS :p.G12S,IFT140:p.E664K,INPPL1:p.T493M,ITGA10:p.R669Q,ITGB1:p.D158N,KIAA1429:p.D1526N,KIAA1429:p.S138F,KPRP:p.E553fs,KSR2:p.T5 55M,LINGO2:p.P410T,LPCAT1:p.V187del,MAGEB3:p.V75A,MAP3K7:p.E524Q,MAP4K3:p.P657fs,MAP9:p.K485N,MARS2:p.R481Q,MBOAT7:p.R424W,MU C16:p.R12774H,MUC5B:p.T4388M,MYH11:p.E993K,MYOCD:p.T493M,MYOM1:p.R63Q,NANOS3:p.S183L,NCOR1:p.R1561Q,NCOR1:p.Q169E,NCR1:p.D213 N,NFE2L2:p.E79K,ODZ1:p.R366M,OPN1MW:p.A285T,OR2M2:p.A95fs,OR2M3:p.M273I,OR2T33:p.R120S,OR6V1:p.I248fs,PABPC5:p.P58L,PACSIN1:p.E359K,PIK3CA:p.M1043V,PIK3CA:p.H1047L,PIWIL1:p.V699M,PLIN5:p.430_431insNG,PLXNA3:p.P5 8S,PRB1:p.R274fs,PRSS1:p.D107N,RAC1:p.A159V,RGS7:p.L21fs,RPA1:p.R31H,RPL18:p.R178fs,S FI1:p.R821Q,SLC35D3:p.*417S,SLC5A7:p.G336C,SMARCA4:p.P913L,STAT3:p.D661V,SYCP2:p.K474 N,SYT6:p.R249H,TBX21:p.E494K,THSD7A:p.R1046C,THSD7A:p.C728F,TMC3:p.R934S,TMTC2:p.T409 R,TP53:p.E285K,TP53:p.C275F,TP53:p.R273C,TP53:p.G266E,TP53:p.G262V,TP53:p.R249S,TP53: p.G245V,TP53:p.C238F,TP53:p.M237I,TP53:p.Y236C,TP53:p.Y236D,TP53:p.R196P,TP53:p.PHHER This includes any combination of mutations selected from the group consisting of C177del, TP53:p.V173L, TP53:p.V173M, TP53:p.Y163C, TP53:p.P151T, TP53:p.V143M, TP53:p.P58fs, URI1:p.S13fs, ZNF177:p.K384N, ZNF750:p.S96fs, and ZZZ3:p.R5Q. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0101] 51. (a) The above group of subjects is suffering from KIRC, (b) At least one of the above tumor-specific mutations is WASH3P:p.G175S, VHL:p.L89H, VHL:p.S111N, WDR52:p.V1227G, KRT1:p.552_559YGSGGSSY>Y, KRTAP1-1:p.S34C, PALM2-AKAP2:p.1075_1076insEA, ZNF814:p.D40 4E,DOPEY2:p.Y2048S,KAT2B:p.W111fs,PABPC1:p.E156fs,PCDHGC5:p.G599V,PIK3CA:p.E545K,RRAD:p.A278E,SIRPA:p.D131del,UQCRFS1:p.I83 V,VHL:p.P45L,VHL:p.V74D,VHL:p.R82P,VHL:p.L116fs,VHL:p.L158V,VHL:p.L169P,WDR73:p.DGTRSQ315del,ABCA3:p.E95D,ABCC5:p.L1090fs,AC ADS:p.R330H,ACAN:p.G952E,ACSM2A:p.L402fs,ADAM23:p.K380M,ADH1A:p.D154V,AFF3:p.SA620del,AGAP6:p.D69fs,AGAP7:p.E71fs,AHNAK:p.5_ 6insE,AIDA:p.K247M,ALAS1:p.G302R,ANAPC16:p.R95fs,ANK2:p.N453S,ANKRD36:p.K378R,ARHGEF5:p.E487G,ARSD:p.AGV234del,ARSD:p.A234G, ATP2A1:p.G704C,ATP7A:p.Q990fs,AVIL:p.G299fs,AXDND1:p.EQ991del,BAP1:p.N78S,BAP1:p.M1I,BLM:p.H660Q,BMPER:p.RIAL444del,BRK1:p. K70Q,BTRC:p.I416M,C16orf55:p.D118A,C19orf33:p.K102E,C20orf132:p.E382D,C2orf71:p.1225_1226insS,C6orf132:p.173_182PPPLLLEPPP>P ,CASP5:p.R23fs,CATSPER4:p.T425M,CCDC120:p.I8V,CCR5:p.S185I,CCZ1:p.E214D,CD7:p.P174fs,CDAN1:p.L646fs,CDH23:p.F1132Y,CDK5RAP2: p.H1592Q,CENPB:p.E410V,CERCAM:p.A85fs,CHEK2:p.K373E,CHIT1:p.P284fs,CLCN2:p.645_645R>RR,CLUL1:p.G463R,CNTNAP4:p.Y436S,CUL9:p.D1726E,CWC25:p.K364E,CXorf51B:p.V43I,DDX39B:p.F149fs,DIRAS1:p.G79C,DISP2:p.F1021S,DNMBP:p.T78P,DOCK8:p.A177fs,DPCR1:p.H383N,DPCR1:p.L768del,EGFR:p.L838M,ENPEP:p.F289C,ESPNP:p.W122fs,FAM105A:p.H126N,FAM186A:p.IPPQAQELEIPL1556del,FAM194B:p.EEEEYL135del,FAM22F:p.S691del,FAM22F:p.P690fs,FAM47A:p.LRPEPPETGVSH235del,FAM47C:p.P388S,FAM78A:p.W192L,FBXO34:p.Q294fs,FGFR3:p.R571fs,FGFR3:p.P716H,FMN2:p.AIPPPPPLPGA956del,FOXD4L4:p.C405fs,FUT6:p.S140fs,GJA1:p.A311fs,GOLGA5:p.L492I,GPM6A:p.A50V,GPRIN1:p.231_239RKEDPGSLR>R,GRAMD1B:p.P356H,GREB1:p.S344Y,GRM6:p.A718fs,GUSB:p.L501V,GUSB:p.C500R,HBB:p.F86C,HDAC6:p.G977D,HEXDC:p.T482P,HNF1B:p.N302K,HNRPLL:p.M327V,HRC:p.P439fs,HSFX2:p.D92E,IL1RAP:p.F50C,IVL:p.EQQEGQLKHP167del,KANK4:p.S253P,KCNJ18:p.E378K,KIAA1751:p.K97N,KRT1:p.SSYGSGG557del,KRT2:p.L299W,KRT4:p.F154fs,KRTAP10-6:p.49_49P>PSCCAP,KRTAP5-7:p.C120Y,KRTAP9-2:p.CCQP140del,LARS:p.P185fs,LCP1:p.P445fs,LOC338651:p.PHRSHSPPWS102del,LRCH2:p.D717G,LTA4H:p.F107L,LYST:p.Q710H,MAFA:p.207_208HH>H,MAGEC1:p.P239del,MAP2. K5:p.Q445R,MAPKAPK2:p.T214fs,MARCKS:p.K152fs,MED12L:p.P2071S,MEGF6:p.A582fs,MGST3:p.G143fs,MLXIPL:p.S790R,MOCOS:p.S849P,MST 1R:p.M464V,MTOR:p.C1483F,MTOR:p.L1460P,MUC16:p.P11260A,MUC17:p.R1227fs,MUC17:p.H1228fs,MUC2:p.1480_1481insI,MUC6:p.P1569fs,M YO3A:p.N525S,NBPF3:p.D491V,NCOR1P1:p.L52P,NDUFA4L2:p.G3fs,NEFH:p.651_651K>CAKSPEK,NES:p.V611L,NFAT5:p.Q906E,NOXO1:p.G3fs,NO 2C1:p.S270I,NSMCE2:p.Q31fs,NUDT21:p.W13fs,ODZ2:p.W628fs,ONECUT1:p.L424M,OR10A3:p.F73V,OR4F4:p.E15G,OR4N2:p.L150fs,OR51B5:p.A 66fs,OR7C1:p.F104fs,PABPC1:p.Y408F,PABPC1:p.K333fs,PABPC1:p.A181T,PABPC3:p.P191T,PALLD:p.A996T,PALM2-AKAP2:p.G1118fs,PARD6A :p.G84fs,PASK:p.T62I,PCDH15:p.C1713F,PCNT:p.G136S,PGM5:p.G426fs,PGPEP1L:p.R164fs,PIK3C2B:p.F1473L,PIK3CA:p.N1044K,PIK3R5:p.L 371R,PITRM1:p.P816T,PLIN4:p.T347I,PODXL:p.28_30PSP>P,POLR1C:p.K332Q,POTED:p.I214V,PPM1E:p.R311W,PRKCE:p.Q157fs,PROX1:p.V225 D,PRRC2C:p.P1883T,PRX:p.P549L,PSD3:p.T563P,PTCH1:p.P689H,RANBP3:p.L386W,RASGEF1C:p.A188T,RGPD6:p.F946L,RHEB:p.Y35N,RIMBP3:p.A396del,RIN3:p.L449V,RLIM:p.S501L,RNF17:p.S351C,RUNX2:p.P466H,SCAF1:p.P208fs,SDK1:p.K508fs,SECISBP2:p.D608E,SERPINB3:p.S209C,SESTD1:p.I306M,SFRP4:p.P325fs,SH3KBP1:p.P563fs,SIPA1L3:p.G777A,SLC13A2:p.L493fs,SLC16A9:p.CVLLGG470del,SLC25A5:p.A118T,SLC44A5:p.V70F,SLC4A8:p.N229K,SLC52A1:p.G370del,SLC52A2:p.G399fs,SLC6A10P:p.K88N,SLC6A14:p.A85fs,SLC9B1:p.V446fs,SON:p.VLESSAVT1359del,SP8:p.G165del,SPAG1:p.353_354insD,SPATA9:p.C189F,SPEG:p.A992fs,SPTB:p.T1864I,SRA1:p.V110L,STAT6:p.P354fs,STK11IP:p.A155E,STXBP3:p.E279G,SVIL:p.M93T,SYNE1:p.R8468S,SYNJ2:p.K832T,SYNPO:p.G619fs,TAOK2:p.Q899fs,TAS2R38:p.I311T,TBC1D12:p.F608Y,TBC1D1:p.H277R,TBC1D3:p.A556fs,TBC1D3C:p.A495fs,TBC1D3F:p.A556fs,TCF7:p.H140P,TDRD10:p.W276C,THRAP3:p.K551R,TMEM102:p.A110P,TMEM161B:p.L142P,TMEM230:p.D140G,TMEM47:p.G87S,TRDN:p.*730Y,TTBK1:p.T1065S,UBE2O:p.R1118fs,UBR5:p.T1306fs,UPK3A:p.G272fs,VHL:p.G39S,VHL:p.S65L,VHL:p.N78D,VHL:p.R79P,VHL:p.W88L,VHL:p.L89P,VHL:p.R107P,VHL:p.S111R,VHL:p.H115N,VHL:p.D121Y,VHL:p.G123fs,VHL:p.D126fs, VHL:p.L12. 8H,VHL:p.L135F,VHL:p.I151T,VHL:p.L153P,VHL:p.L158P,VHL:p.Q164fs,VHL:p.L184P,VHL:p.L188 P,WASH6P:p.315_316insAPP,WASH6P:p.T201M,WWP2:p.G458A,ZCCHC6:p.K937N,ZFAND2B:p.I149T,ZFR This includes any combination of mutations selected from the group consisting of 2:p.Y107N, ZNF273:p.N319K, ZNF462:p.S650T, ZNF516:p.A256D, ZNF519:p.H431Y, ZNF687:p.F858C, ZNF732:p.E227Q, ZNF880:p.Q406R, ZP3:p.V362fs, and ZRANB1:p.*735fs. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0102] 52.(a) The above group of subjects is suffering from LAML, (b) At least one of the above tumor-specific mutations is: NPM1:p.W288fs,DNMT3A:p.R882H,NPM1:p.L287fs,IDH2:p.R140Q,IDH1:p.R132C,FLT3:p.D835Y,DNMT3A:p.R882C,FLT3:p.600_601insFREYEYD,IDH1:p.R132H,NRAS:p.G13D,U2AF1:p.S34F,KIT:p.D816V,FLT3:p.D835E,IDH2: This includes any combination of mutations selected from the group consisting of p.R172K, NRAS:p.G12D, WT1:p.S381fs, ABTB1:p.L249fs, DNMT3A:p.R736H, FLT3:p.D835H, KRAS:p.G12D, NPM1:p.L287fs, NRAS:p.Q61H, NRAS:p.Q61K, PHACTR1:p.V251fs, RBBP4:p.E330K, RUNX1:p.R135G, and U2AF1:p.S34Y. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0103] 53.(a) The wavelength of a stirrup is determined by LUAD. (b) Indicated are the constructs of 1 and KRAS:p.G12C,KRAS:p.G12V,EGFR:p.L858 R,U2AF1:p.S34F,KRAS:p.G12A,TP53:p.R158L,KRAS:p.G12D,PIK3CA:p .E545K,TP53:p.R273L,EGFR:p.ELREA746del,KRAS:p.G13D,A2ML1:p.S654fs,BRAF:p.G469V,CTNNB1:p.S37F,EGFR:p.G719A,KRAS:p.G13C,MY OF:p.G165fs,EGFR:p.S768I,FAM47C:p.G948W,KRAS:p.Q61L,MYH10:p.L1091fs,NRAS:p.Q61L,OR4C3:p.H130fs,PI15:p.V22F,RAD50:p.D69Y, RIT1:p.M90I,TP53:p.C275F,TP53:p.R249M,TP53:p.R249G,TP53:p.R248P,TP53:p.R175H,TP53:p.Y163C,TP53:p.A159P,TP53:p.V157F,TP53 :p.G154V,ABCB1:p.R467L,ACBD3:p.R224L,ACTA1:p.G275C,ACTN2:p.D893Y,ADAM30:p.Q741H,ADAMTS14:p.G238C,ADAMTS20:p.R1251S,ADAMT S20:p.R541L,ADAMTS5:p.L549M,ADAMTS9:p.G659W,ADCY2:p.P1016T,ADCY5:p.G623C,AFP:p.A182G,AHDC1:p.P155Q,AKAP1:p.LDRNEEG317del ,ALKBH1:p.K137E,ANK2:p.Q3076L,ANKRD44:p.G339C,ANO3:p.A41S,AP1G1:p.R723L,APBB2:p.T243fs,APOB:p.L973M,APOBR:p.R840L,AQP10: p.Q261L,ARAP3:p.R1226L,ARFIP2:p.R86L,ARHGAP36:p.P16H,ARL13B:p.R358L,ASCC2:p.R365L,ASPM:p.S240F,ASXL3:p.P1470Q,ATRN:p.P197Q,AVIL:p.G64W,AXDND1:p.W101R,B3GAT1:p.R125L,BARX2:p.R68P,BCL9L:p.G980C,BCOR:p.N1459S,BEND2:p.P536Q,BMS1:p.GAF55,VBR45:p.V. 0E,BRAF:p.G466V,BRD9:p.G330W,BRF1:p.V469L,BRWD3:p.H160N,BTRC:p.G260W,C11orf68:p.V135L,C15orf2:p.V753F,C15orf2:p.G330W,C15orf2:p.C196 8:p.M61I,C1GALT1:p.G299V,C1orf173:p.G1454S,C1orf173:p.S688Y,C1 orf87:p.R541L,C2orf53:p.P272H,C3orf20:p.R740L,C7:p.R687S,C7orf 58:p.G140W,C7orf58:p.R238L,CACNA1A:p.S772Y,CACNA1D:p.R1073L,CACNA1E:p.R2089Q,CACNA2D1:p.A352E,CACNG3:p.R23PS,C959:C ALB2:p.R258C,CAMK2B:p.G131V,CARD11:p.I1065M,CCDC111:p.R417L,CCDC141:p.E1204V,CCDC19:p.R279L,CCDC19:p.R207L,CCKAR:p.CDLC111M, B:p.W41L,CDH10:p.S577R,CDH10:p.R472C,CDH10:p.R128S,CDH18:p.A721S,CDH20:p.P433H,CDH6:p.Q237K,CDK13:p.R880S,CLDK244L,CDH10:p. p.A309P,CFDP1:p.P129fs,CHN1:p.K264N,CHRNA4:p.S396R,CHRNA9:p.P361Q,CLCNKA:p.P124Q,CLEC12B:p.W217L,CLK4:p.R68L,CNTFR5:p.D22Y,CY NTN6:p.R807M,CNTNAP2:p.F395L,COL19A1:p.P538Q,COL5A2:p.G612W,COL5A2:p.G516W,COL9A1:p.P211Q,CPE:p.P290Q,CPNE8:p.Q14:p.CPH.P219Q,CRIPAK:p.S180fs,CROT:p.Q580H,CRTC3:p.S363L,CSMD2:p.P1855Q,CSMD3:p.T2810N,CSMD3:p.P2727T,CSMD3:p.Q174H,CUBN:p.G596C,CUL4B:p.R91S,CUL7:p.L371F,CXCL9:p.K122N,CXCR4:p.E345Q,CXorf59:p.R198M,CYP11B1:p.R498G,CYP27A1:p.P112Q,CYP2B6:p.A444E,DACH2:p.R539L,DCC:p.R446H,DDX56:p.R329L,DEFA1:p.W90C,DENND2A:p.R688Q,DENND2A:p.R499L,DMBT1:p.R1521L,DNAH5:p.R3822L,DNAH9:p.S2993R,DNAI2:p.V231L,DPP6:p.L757F,DSG4:p.R128L,DST:p.A4410S,DZIP3:p.M322L,EBF3:p.R231S,EFCAB4B:p.E265Q,EHHADH:p.Q704H,ELAVL2:p.L263F,EMR1:p.R493H,ENAH:p.R514L,ENPP1:p.G738E,EPB41L3:p.A896S,EPG5:p.R2289L,EPHA1:p.G111V,EPHB6:p.R337H,EPRS:p.V1151L,ERBB2:p.S310Y,ERBB2:p.774_775insAYVM,ERBB2:p.776_776G>VC,ERN2:p.T295K,FAM120B:p.P467H,FAM127C:p.F52L,FAM135B:p.W240C,FAM210B:p.L112F,FAM47A:p.R690L,FAM47B:p.W163C,FAM47B:p.L567F,FAM5C:p.R457G,FAM70B:p.P277T,FAM71B:p.L583M,FAM75A6:p.R304S,FAM75A6:p.P54L,FAM75D1:p.R1265S,FARP1:p.R299L,FAT1:p.R4359L,FAT3:p.R1266H,FAT3:p.G1899V,FAT3:p.H3574N,FBXO18:p.M144I,FBXO31:p.G443fs,FCGBP:p.A1022S,FCRL2:p.V505L,FER. D3L:p.P92H,FGB:p.E339Q,FGFR2:p.E116K,FGFRL1:p.R243L,FGFRL1:p.V274L,FKBPL:p.R320L,FLG2:p.G1545V,FLG2:p.L572F,FLG:p.P3254H,FLG:p.P2466Q,FMN2:p.P992T,FOLH1:p.A643S,FOXRED1:p.R136L,FRAS1:p.C382F,FRG2B:p.D142Y,FRMPD1:p.E1093Q,FSHB:p.T43N,GABRA5:p.Q224K,GADL1:p.L352I,GAL3ST3:p.A271S,GALNT14:p.D234E,GAS8:p.R313S,GATA3:p.M443I,GCDH:p.R82C,GEM:p.R268L,GFRAL:p.Q308K,GIT2:p.R123L,GJB4:p.R22S,GLB1L2:p.I407M,GLOD4:p.Q223fs,GNAO1:p.P283Q,GPNMB:p.I174M,GPR137B:p.G240C,GPR158:p.P762T,GPR98:p.G4307W,GRB7:p.R239L,GRHL1:p.G608W,GRID1:p.R683L,GRIK1:p.R368Q,GRM5:p.P895fs,GTF2E1:p.R192L,H3F3C:p.R131L,HAO2:p.H12N,HCN1:p.P231Q,HECW1:p.A183S,HGF:p.M686T,HIP1:p.R940L,HIST1H1E:p.R25P,HLA-DMA:p.A236fs,HOXA5:p.G11C,HS3ST3A1:p.G399W,HSD17B6:p.F209L,HSPA13:p.V85L,HSPBAP1:p.R282L,HTR5A:p.W298C,IGHMBP2:p.R615S,IL2:p.R103M,IL2RA:p.G61W,IL32:p.P215T,ING1:p.A220S,INMT:p.G56V,ITGA8:p.G616C,ITGAD:p.L528fs,ITGAX:p.R283H,ITIH1:p.G254W,ITIH2:p.L842V,ITK:p.R29L,ITPR2:p.P358Q,JMJD1C:p.R1198S,KCNA1:p.G376C,KCNH8:p.M455I,KCNJ3:p.L430F,KCNK18:p.G23V,KCNK2:p.R166L,KEAP1:p.G603W,KEAP1:p.R260L,KEAP1:p.S144F,KHDRBS2:p.S203L,KIAA1211:p.P1203Q,KIAA1549:p.L1272F,KIAA1755:p.Q108H,KIF15:p.E252Q,KIF9:p.G480R,KIRREL:p.G604C,KLF5:p.E419Q,KRAS:p.Q61H,KRTAP10-12:p.R64P,KRTAP27-1:p.M124I,KRTAP4-5:p.C91F,KRTAP5-1:p.S193Y,L1CAM:p.R632S,L3MBTL4:p.W162L,LAMA1:p.D1030Y,LAMB1:p.T1610fs,LAMB4:p.G1239W,LAMB4:p.G588W,LEF1:p.I53V,LEKR1:p.Q450K,LIM2:p.S150T,LIPJ:p.P236Q,LPHN3:p.E740D,LPPR4:p.R527S,LRFN5:p.N132K,LRP1B:p.G3563C,LRP2:p.M4039I,LRRC4C:p.Q10L,LRRIQ1:p.W792L,LRRTM4:p.S243Y,MAGEA10:p.R7H,MAGEC2:p.W109C,MAGI1:p.G1156V,MAGI2:p.P1044T,MAK:p.P373Q,MAP2K1:p.K57N,MARCH11:p.R193L,MEPE:p.G142C,MKI67:p.R1081S,MKRN3:p.P448H,MLL3:p.N393K,MLL3:p.Q356K,MMRN1:p.A1013S,MOGAT2:p.Q66fs,MXRA5:p.D324Y,MYH4:p.T790M,MYH8:p.R1117C,MYH8:p.H1006N,MYO5B:p.R708L,MYO7B:p.P2040H,MYO9B:p.R94L,MYT1L:p.P351Q,NAA11:p.T184K,NAB1:p.L72F,NAV1:p.R938L,NBPF15:p.G665E,NCAM2:p.G698C,NCAPD2:p.R220L,NDST3:p.V427I,NEK2:p.R239S,NFIA:p.L294F,NLRP3:p.R157C,NOTCH2:p.R2105L. ,NR4A2:p.R314L,NRG1:p.V481L,NRXN1:p.R813S,NRXN1:p.A660S,NRXN3:p.P23H,NRXN3:p.R103C,NTM:p.G333C,NUAK1:p.G173C,NYAP2:p.P437L,ODZ3:p.P218Q,OIT3:p.R508S,OOEP:p.R101C ,OPN1LW:p.P283H,OR10H4:p.M199I,OR10J1:p.L157Q,OR10X1:p.L298I,OR10Z1:p.L205F,OR14A16:p.G160C,OR2A25:p.M80I,OR2AG2:p.G249W,OR2AK2:p.W37C,OR2H2:p.L205F,OR2J2:p.G234W,OR2L13:p.M106I,OR2L13:p.T242A,OR2L3:p.M1I,OR2L3:p.L67I,OR2L8:p.R121C,OR2L8:p.R171S,OR2M2:p.F177L,OR2M2:p.F323L,OR2M5:p.V205L,OR2T12:p.M258L,OR2T27:p.D11Y,OR2T33:p.P165Q,OR2T34:p.C246F,OR2T6:p.V213L,OR4C12:p.D309Y,OR4C12:p.M279I,OR4C16:p.L162M,OR4M2:p.A119S,OR4M2:p.A161S,OR51V1:p.P298T,OR5AS1:p.M39I,OR5B12:p.S289C,OR5B17:p.M266I,OR5D14:p.H246N,OR5D16:p.P264T,OR5D18:p.R123H,OR5F1:p.G44V,OR5J2:p.A36S,OR5L1:p.T275N,OR6C65:p.I154fs,OR6C75:p.G94W,OR6K2:p.P79Q,OR8D2:p.R306M,OR9A2:p.R289W,OR9G9:p.R169L,P2RX7:p.P142Q,P2RY10:p.T10K,P2RY10:p.V196L,PABPC5:p.R99S,PAPPA2:p.P917T,PAPPA2:p.P1706H,PBLD:p.P55Q,PCDH10:p.R587S,PCDH10:p.V986L,PCDH11X:p.R1010I,PCDHAC2:p.A742V,PCDHB5:p.P649S,PCDHGC5:p.K12N,PCDHGC5:p.P684H,PCLO:p.P3946T,PCMTD1:p.R271M,PDPR:p.G793W,PDYN:p.G191W,PDZD2:p.R565S,PDZD8:p.S980G,PFKM:p.R118S,PIGM:p.R225L,PIK3CA:p.E542K,PIK3CG:p.V165I,PILRA:p.S291fs,PLCE1:p.G564C,PLCL1:p.M564I,PLEKHA6:p.R110L,PNKP:p.G174W,POGZ:p.G75W,POLE:p.R573L,POM121L12:p.P231T,POM121L12:p.P242H,POTEE:p.V288M,POTEM:p.S78R,POU3F3:p.D321Y,PPT2:p.R265L,PRDM16:p.P1036L,PRELP:p.D201Y,PRPF40B:p.R160S,PRPF6:p.R763L,PTEN:p.R234L,PTPN11:p.G503V,PTPN13:p.E2067K,PTPRJ:p.G334W,PTPRT:p.R928L,PTPRU:p.P559S,PXDNL:p.P1456T,QSOX1:p.R401L,QSOX2:p.R683L,RAB13:p.R167L,RAB8A:p.G20W,RAPGEFL1:p.R356L,RBM19:p.G390W,RCL1:p.P112Q,REG1B:p.W57L,REG3A:p.S150L,REG4:p.G110V,RIMS2:p.R55L,RIT2:p.R85L,RLN2:p.S138C,RNF20:p.P529Q,RORB:p.G94W,RPL10L:p.K187T,RPRD2:p.R97S,RTN1:p.S103W,RUNX2:p.R337M,RYR2:p.K2413N,RYR2:p.M4334I,RYR3:p.P1670T,S100PBP:p.R5L,S1PR1:p.L104F,SAGE1:p.H298Q,SALL1:p.E965K,SALL1:p.R898W,SALL4:p.R187L,SBSPON:p.G133W,SCAF8:p.G740C,SCG2:p.P252Q,SCML4:p.L261F,. SCN2A:p.T155K,SEC24D:p.A50fs,SEC61A2:p.G126V,SERPINA12:p.D253Y,SERPINA9:p.M414I,SERPINC1:p.R45L,SGIP1:p.R502L,SH3GL3:p.R174L,SH3PXD2A:p.S759L,SI:p.V1217F,SKOR1:p.Y883C,SLC1A2:p.F348fs,SLC24A5:p.R35S,SLC25A48:p.R101S,SLC35E2:p.R201L,SLC39A12:p.C628S,SLC39A6:p.R53L,SLC4A5:p.I533V,SLC5A1:p.G53W,SLC5A7:p.G442V,SLC6A11:p.W299L,SLC6A2:p.S354C,SLC8A1:p.G433C,SLIT1:p.R1460L,SLITRK5:p.R68L,SLITRK5:p.R468M,SLITRK6:p.N741K,SORL1:p.R205L,SOS1:p.N233Y,SOX9:p.E75K,SPAG16:p.V439L,SPIN4:p.Y171C,SPRR2D:p.P30fs,SPTA1:p.G2367C,SPTA1:p.D2243Y,SSX3:p.P127T,ST18:p.H778Q,STAC3:p.G117W,STOML3:p.D86Y,STX2:p.R107L,SUMF2:p.G110E,SUN3:p.P339Q,SV2C:p.P60Q,SYNDIG1:p.D135Y,SYNE1:p.K8632E,TARS2:p.E199K,TAS2R16:p.Q177H,TCOF1:p.K264R,TCTE1:p.S127I,TDO2:p.Q197H,THSD7A:p.G810W,THSD7A:p.R801L,TIFAB:p.D43E,TIGD4:p.S312F,TLL1:p.P53Q,TMPRSS11E:p.G259C,TMTC1:p.A864D,TMTC1:p.G212V,TMX3:p.R151C,TNNI1:p.R67L,TNR:p.L692I,TOP2A:p.R736L,TP53:p.R337L,TP53:p.E285K,TP53:p.R283P,TP53:p.D281N,TP53:p.C277F,TP53:p.V274F,TP53:p.R273H,TP53:p.I255F,TP53:p.R249S,TP53:p.M237I,TP53:p.S215I,TP53:p.C176F,TP53:p.R110L,TP53:p.G105C,TP53:p.P72fs,T PO:p.E558K,TRAF6:p.R502S,TRIM42:p.Q127K,TRIM48:p.A93D,TRIM4:p.R398L,TRIM51:p.W131C,TRIM9:p.R337S,TRIML1:p.H399Q ,TRPM3:p.G298W,TSC1:p.G378C,TSG101:p.R276S,TSHZ1:p.K501N,TSHZ3:p.G677V,TTF2:p.R761S,TUBA3C:p.Q176fs,UBAC1:p.K33 0N,UBE2J2:p.G193W,UBR1:p.G1647W,UGT2B7:p.M214I,VMP1:p.E369Q,VPS13B:p.G2575W,VSTM2A:p.G75V,VWA3B:p.R557L,WBP11:p. P227fs,WDR52:p.G612C,WDR59:p.R837S,WDR75:p.P287Q,WDR88:p.G100W,ZCCHC5:p.G335W,ZFHX4:p.L811F,ZFHX4:p.T1663N,ZFHX 4:p.H2511Q,ZFP14:p.Q17L,ZIC1:p.A112E,ZNF154:p.T408N,ZNF223:p.G23W,ZNF295:p.S732C,ZNF322:p.K106N,ZNF385D:p.T226S, This includes any combination of mutations selected from the group consisting of ZNF454:p.S190I, ZNF492:p.P392H, ZNF521:p.G640C, ZNF521:p.P270H, ZNF536:p.G186C, ZNF536:p.G663W, ZNF644:p.G21W, ZNF716:p.H263L, ZNF71:p.V411L, ZNF782:p.G484W, ZNF831:p.Q617K, ZNF98:p.C492F, and ZSWIM2:p.S214Y. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0104] 54. (a) The above group of subjects suffers from LUSC,(b) Structural structures of PIK3CA:p.E545K,TP53:p.R158L,KRTAP5-5:p .GCG47del,NFE2L2:p.E79Q,CDKN2A:p.D108Y,DHX9:p.V40G,MAFA:p.207_ 208HH>H,NFE2L2:p.R34Q,PBX2:p.Y262F,PIK3CA:p.E542K,TP53:p.R273L ,TP53:p.C242F,TP53:p.R175G,TP53:p.Y163C,TP53:p.V157F,AICDA:p.R1 31G,ALPK2:p.D53N,ANKFN1:p.M280I,ARPC1A:p.F212L,ASXL2:p.S1081L,C1orf74:p.D254N,C3orf30:p.D227E,CCDC121:p.W397L,CHN2:p.I43M,CLE C4C:p.R179L,CLN3:p.G206S,CNTN5:p.T178N,COL12A1:p.G2753C,CPS1:p.T855K,CSMD3:p.T1094K,CSMD3:p.Q691K,DDX11:p.R167T,EGFR:p.L861Q, EME1:p.D570H,EP300:p.D1399N,ESYT3:p.S574F,FAM135B:p.L648M,FAM135B:p.Q285H,FAM47A:p.G372W,FBXW7:p.R505G,FGFR3:p.S249C,GALNT13 :p.G358C,GNL3L:p.K20N,GPC5:p.R347L,HCN1:p.A714S,HCN1:p.R659L,HCN1:p.G499V,HCN1:p.P326T,HERC2P3:p.A803V,HEXDC:p.T482P,HIST1H3B :p.E74K,HIST2H2BE:p.G54D,IFNA10:p.V79A,IL7R:p.S54L,INADL:p.P1340A,ISX:p.C2F,ITGAX:p.R685H,ITPR1:p.E1883Q,KCNN3:p.80_81insQQ,K EAP1:p.G480W,KEAP1:p.R470C,KEAP1:p.V155F,KIAA1751:p.L63F,KIAA2022:p.C345F,KIR3DL2:p.K229E,KLF5:p.E419Q,LAMA4:p.M1293I,LMLN:p.G199C,LRP2:p.A516V,LRRC66:p.F458L,LSG1:p.R517L,LUM:p.R310L,MB21D2:p.Q311E,MCHR1:p.S306F,MKRN3:p.G270V,MUC16:p.N1 1594K,NFE2L2:p.G81S,NFE2L2:p.G31A,NFE2L2:p.L30F,NFE2L2:p.D29H,OR2B11:p.G10V,OR2T2:p.F13V,OR4K2:p.C254F,OR51F2:p.R 67P,OR51S1:p.R159Q,OR5D18:p.T271K,OR8H2:p.L166F,OR8J3:p.S160L,OR8K3:p.K235N,PCDHB1:p.N568K,PHIP:p.I1681M,PIK3CA:p .E726K,PIK3CA:p.H1047R,PLCE1:p.G439C,PRSS57:p.E39Q,PYHIN1:p.G148A,RANBP6:p.I984L,RBMXL1:p.G305C,REG1B:p.M67I,RGS6 :p.W366L,RNF5:p.T136I,RP1:p.S1771L,RRP15:p.L214F,RYR2:p.E711K,SAMD3:p.Q206H,SLITRK3:p.R214L,SON:p.S908L,SP4:p.E11 part,STK11:p.G279fs,TARBP1:p.L782V,TBCD:p.R476C,TMPRSS11F:p.R274Q,TP53:p.R337L,TP53:p.E271K,TP53:p.R267P,TP53:p.G2 45V,TP53:p.Y234C,TP53:p.Y220C,TP53:p.H214R,TP53:p.H193L,TP53:p.H179L,TPTE:p.M541I,TRIM7:p.L332I,TTN:p.T32425M,ZFP 36L2:p.D240N,ZNF208:p.H883Q,ZNF48:p.R235H,ZNF626:p.K473R ,ZNF676:p.P43T,ZZZ3:p.R162Q This is a slightly larger quantity of skeletal muscle. Ranges 30~36 of the range 30~36.

[0105] 55. (a) The above group of subjects is suffering from OV, (b) At least one of the above tumor-specific mutations is TP53:p.R273H, TP53:p.Y220C, TP53:p.R248Q, TP53:p.R175H, TP53:p.R273C, TP53:p.I195T, TP53:p.R248W, TP53:p.R282W, TP53:p.C176Y, TP53:p.V157F, TP53:p.S241F, TP53:p.H179R, TP53:p.G245S, TP53:p. H193R,ADCY2:p.V888I,B2M:p.M1V,BAP1:p.R227C,CYP4A11:p.V185F,DNAH5:p.R3197Q,GART:p.K807fs,GRIN2B:p.R51 9Q,HRNR:p.M1fs,KLHL29:p.L716fs,KRAS:p.G12V,MGA:p.R2435Q,MYO3A:p.N525S,NPAS2:p.Q201R,NRAS:p.Q61R,PDAP 1:p.K55fs,PGAP1:p.F565C,TP53:p.S315fs,TP53:p.C275Y,TP53:p.R273L,TP53:p.V272M,TP53:p.G266V,TP53:p.G26 6R,TP53:p.D259Y,TP53:p.P250L,TP53:p.G245D,TP53:p.G245V,TP53:p.G244C,TP53:p.C238fs,TP53:p.Y236C,TP53: This includes any combination of mutations selected from the group consisting of p.Y234C,TP53:p.V216M,TP53:p.S215R,TP53:p.Y205C,TP53:p.L194R,TP53:p.P191del,TP53:p.Y163C,TP53:p.A159V,TP53:p.K132N,TRPC7:p.D210V,UXS1:p.V100L,WNT11:p.C344Y, and ZNF295:p.E885A. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0106] 56.(a) The above group of subjects suffers from READ, (b) At least one of the above tumor-specific mutations is: KRAS:p.G12V,TP53:p.R273H,KRAS:p.A146T,KRAS:p.G12D,TP53:p.R175H,AKAP9:p.L3482I,APBA1:p.E624K,BAG5:p.D439N,C17orf97:p.E230D,CDH23:p.F177L,CERS3:p.E95D,DNAH5:p.R982H,ERBB2:p.V842I,GABRB3:p.D500N,KRAS:p.G13D,KRAS:p.G12C,KRAS:p.G12S, This includes any combination of mutations selected from the group consisting of LRP6:p.R675Q, MACF1:p.F722L, MBOAT2:p.R43Q, MYO1D:p.E246K, NLRC4:p.E409K, NRAP:p.E327K, NRAS:p.Q61K, PCDH15:p.R1552I, PIK3CA:p.N345K, PIK3CA:p.E545K, POLE:p.S459F, PPP2R2B:p.P326L, SMAD4:p.R361H, TP53:p.R248W, ZFP2:p.R150I, and ZNF563:p.K26N. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0107] 57.(a) The above group of subjects is suffering from SKCM, (b) At least one of the above tumor-specific mutations is BRAF:p.V600E,NRAS:p.Q61R,NRAS:p.Q61K,HSD17B7P2:p.N175S,BRAF:p.V600K,DISP1:p.G732L,IDH1:p.R132C,NRAS:p.Q61L,MUC16:p.P5119S,RAC1:p.P29S,WASH3P:p.G175S,AGAP9: p.M248V,C15orf23:p.S24F,DNAH5:p.D3236N,SPTLC3:p.R97K,TMC5:p.R276C,CFB:p.R314M,FRG1B:p.A50P ,INMT:p.S212F,LOC649330:p.G93E,MAP2K1:p.P124S,RGS7:p.R44C,STK19:p.D89N,ADAM30:p.G97L,ARL16 :p.G6R,ARMC4:p.E22K,BRAF:p.K601E,CAPN13:p.P405S,CD1C:p.R89C,CLCC1:p.P406Q ,CNTN5:p.S379F,DNAH5:p.R742Q,EEF1B2:p.S43G,FRG1B:p.I59V,GABRG1:p.E205K,IAR S2:p.R832C,IL32:p.D218fs,ISX:p.R86C,KLHDC7A:p.E635K,NAP1L4:p.P285Q,NBPF10 :p.Q908E,OR2A5:p.S71L,OR4E2:p.R226Q,OR4M1:p.G41E,OR4M2:p.S268F,OR4N2:p.G41 This includes any combination of mutations selected from the group consisting of E,OR51B2:p.S163L,PCDHGC5:p.R293C,PCLO:p.R4133C,PHGDH:p.G173L,POTEG:p.D51N,PPP6C:p.R301C,PRAMEF11:p.C84S,PSG9:p.E404K,PTPRB:p.D1560N,RNF152:p.P95S,SPAG16:p.P488S,SPATA8:p.E18K,TAF1A:p.R172M,TCEB3C:p.E308K,THSD7B:p.E126K,TTN:p.E12129K,XIRP2:p.D2439N, and ZNF831:p.R1393Q. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0108] 58. (a) The above group of subjects is suffering from UCEC, (b) Structural structures of 1 and 1 are RPL22:p.K15fs,PTEN:p.R130G,PTEN:p.R13 0Q,KRAS:p.G12D,KRAS:p.G12V,PIK3CA:p.H1047R,PIK3CA:p.R88Q,PIK3C A:p.E545K,PTEN:p.V317fs,FGFR2:p.S252W,PIK3CA:p.E542K,CTNNB1:p.S37F,POLE:p.P286R,PPP2R1A:p.P179R,CTNNB1:p.S37C,KRAS:p.G13D,CT NNB1:p.D32N,CTNNB1:p.S33F,CTNNB1:p.G34R,KIAA2026:p.R574C,LIMCH1:p.R806fs,PIK3CA:p.H1047L,ALPK2:p.K523fs,CTNNB1:p.S33C,FBXW7 :p.R505C,HPD:p.R284fs,KRAS:p.G12A,PIK3CA:p.R93Q,POLE:p.V411L,TP53:p.R248W,ABCA11P:p.R385I,ABI1:p.K445N,ACSM2B:p.K195N,APOB:p .F3102L,ASCC3:p.R136Q,C12orf4:p.R335Q,CCDC132:p.R838C,CHD4:p.R975H,CSDE1:p.R220C,CTNNB1:p.D32Y,CTNNB1:p.S33Y,CTNNB1:p.T41I, EXOC1:p.R588C,FBXW7:p.R465H,FGFR2:p.N549K,FUBP1:p.R430C,GEN1:p.S509L,IK:p.E90fs,KIF20B:p.E54K,MAX:p.H28R,MBOAT2:p.R43Q,METTL 14:p.R298P,MFGE8:p.D170N,MS4A8B:p.S3L,NSMCE1:p.D244N,OXR1:p.E122K,PCDH19:p.E530K,PIK3CA:p.R108H,PIK3CA:p.N345K,PIK3CA:p.C420 R,PIK3CA:p.Q546P,PIK3CA:p.Q546R,PTEN:p.R130L,RBL2:p.E127K,RXFP1:p.S223Y,SF3B1:p.R957Q,SLC20A1:p.P328fs,SOX17:p.S403I,TNS1:p.Q659del,TP53:p.R273H,TP53:p.R273C,TP53:p.R248Q,TTN:p.D16823N,TXNL1:p.R234C,ZFHX3:p.R1893fs,ZNF180:p.R625I,ZNF257:p.R392I,ZNF354B:p.D609N,ZNF43:p.R280C,ZNF709:p.R468I,ZNF765:p.S254L,ABCA5:p.R1476Q,ACVR1:p.R206H,ADAD1:p.S11L,ADAM9:p.R256Q,ADD3:p.E570K,ADGB:p.S1124L,AGXT2:p.R502C,AMBN:p.S225Y. ,ANKDD1A:p.R24H,ARHGEF33:p.R46I,ATP10B:p.L1304I,ATP2C1:p.E724K,ATP9A:p.R290Q,ATR:p.R1814fs,AVL9:p.F34L,BMPER:p.R241Q,BTN3A2:p.E153K,C14orf118:p.R279I,C14orf166B:p.F230L,C3orf23:p.R217C,C3orf62:p.R185Q,CACNA1C:p.S710L,CAGE1:p.E539K,CARD10:p.KE272del,CCDC144A:p.S1264L,CCDC168:p.D5020Y,CCDC36:p.R209I,CD55:p.E156K,CEP44:p.S253L,CIITA:p.E728K,CREBBP:p.P2094L,CTNNB1:p.S37A,CTTNBP2:p.S420L,DCT:p.R532Q,DIAPH2:p.E121K,DLG2:p.S624L,DNAH10:p.R1888Q,DNAH14:p.R1367C,DNAH7:p.R2961Q,DNAH8:p.R1347H,DNAJC13:p.E1248K,DNMT1:p.E51K,DST:p.S1767Y,DYNC2H1:p.E883D,EMR1:p.R631Q,EPHX4:p.R282Q,ERCC6L2:p.L445I,F10:p.E117K,FAM155B:p.E158K,FAM83B:p.R206Q,FARP1:p.S383L,FAT3:p.A4159T,FBXW7:p.R689W,FBXW7:p.R465C,FBXW7:p.G423V,FN1:p.R290C,FZD6:p.R416Q,GABRA3:p.R73H,GABRA4:p.R460Q,GALNTL2:p.E395K,GFAP:p.A233T,GGA2:p.A63V,GIGYF2:p.R227H,GNPTAB:p.R1189Q,GPR112:p.S1283Y,GPR98:p.R4142W,GRIA3:p.S646Y,GRM6:p.E363D,HMCN1:p.S133Y,HSPA4L:p.R483C,HTR2A:p.S219L,INTS7:p.R940C,INTS7:p.R106I,ITM2C:p.E167K,JAKMIP2:p.R283I,KCND3:p.S438L,KCNS2:p.D211N,KDM1B:p.F361L,KIAA0556:p.L330I,KIAA1147:p.A149V,KIF23:p.R150Q,KIF27:p.K925N,KIF9:p.R594Q,KLHL13:p.E213K,KLHL28:p.E33K,LIN9:p.R183W,LRBA:p.E2103K,LRP2:p.R2432I,MAGI2:p.L450M,MC5R:p.A109T,MEGF10:p.S1053L,MKI67:p.T1664fs,MKLN1:p.F485L,MMRN1:p.F917L,MSH4:p.E730K,MTOR:p.S2215Y,MUC7:p.S336L,MYBPC2:p.R646H,N4BP2L2:p.R506C,NAPSA:p.R121Q,NCOA7:p.E369D,NCR1:p.R258W,NEK11:p.R374Q,NHEJ1:p.R109Q,NNMT:p.E233K,NOTCH4:p.15_16LL>L,NPY1R:p.A371T,NRAS:p.Q61R,OGDHL:p.R57C,OMA1:p.R445Q,OPRM1:p.R462C,OR4C12:p.F248L,OR5AK2:p.K89N,OSBPL6:p.R577Q,PCDHAC2:p.K138N,PCDHB12:p.R289C,PCDHGC5:p.A70T,PIK3CA:p.R38H,PIK3CA:p.E39K,PIK3CA:p.E110del,PIK3CA:p.K111E,PIK3CA:p.Q546K,PIK3CA:p.M1043V,PIK3CA:p.M1043I,PLA2G3:p.R201Q,PLXNA1:p.E1295K,PON1:p.R306Q,POTEE:p.R303I,POTEF:p.K674N,PPP2R1A:p.S256F,PPP2R3B:p.F310L,PRAM1:p.A268T,PREX1:p.E1246K,PRKCQ:p.A324V,PTEN:p.R130P,PVRL4:p.A358T,RAI2:p.S385Y,RBM39:p.T353I,RELN:p.F2722L,RFPL1:p.R148Q,ROBO2:p.D1018N,ROS1:p.R245I,RPS6KA6:p.S394Y,RSBN1:p.E572K,RYR1:p.A2576T,S. ACS:p.R2906Q,SCAPER:p.R366Q,SELP:p.R429W,SENP7:p.S673Y,SEPHS1:p.E13K,SFRP4:p.R232Q,SGK1:p.K367del,SIX1:p.E191K,SLC10A7:p.S261L,SLC12A2:p.R828Q,SLC16A14:p.R495Q,SLC7A2:p.R322W,SMCR8:p.E175K,SOS1:p.N233Y,SPOP:p.E50K,STRN3:p.K218N,STXBP6:p.D92N,SULT1E1:p.R77Q,SUN3:p.L124I,SUSD1:p.R343C,SYNM:p.R516Q,TAF1:p.R843W,TDRD3:p.R322Q,THADA:p.S1941L,TLN2:p.S208L,TMEM161B:p.R315Q,TMPRSS3:p.R16Q,TP53:p.Y220C,TPTE:p.S423L,TRANK1:p.E846K,TRPC5:p.S490L,TRPM3:p.R429W,TSSK1B:p.E301K,TTLL7:p.R751H,TTN:p.S20317L,TTN:p.E6404K,TTN:p.R4434Q,TTN:p.R2506Q,UGT8:p.E102K,USF1:p.R52Q,USP16:p.R455Q,USP25:p.R873H,USP33:p.R36Q,VPRBP:p.R802Q,VPS13B:p.R692Q,WDR65:p.F110C,YTHDC2:p.E185K,ZFYVE1:p.R266Q,ZKSCAN1:p.R541fs,ZNF117:p.R157I,ZNF180:p.R569I,ZNF195:p.R59Q,ZNF254:p.K179N,ZNF263:p.R510I,ZNF333:p.R554Q,ZNF354B:p.R402I,ZNF442:p.R309Q,ZNF454:p.R376I,ZNF485:p.R374I,ZNF488:p.R206Q,ZNF559:p.E284K,ZNF594:p.R287I,ZNF611:p.R390I,ZNF645:p.R154C,ZNF649:p.R338Q,ZNF649:p.R198I,ZNF674:p.R405I,ZNF675:p.R220I,ZNF678:p.R564I,ZNF732:p.This includes any combination of mutations selected from the group consisting of R354I, ZNF780A:p.R466Q, ZNF823:p.R547I, ZNF836:p.R854I, ZNF836:p.R630I, ZNF841:p.R757I, and ZNF98:p.R370I. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0109] 59. (a) The above group of subjects is suffering from ACC, (b) At least one of the above tumor-specific mutations is ZFPM1:p.EPL444del, GARS:p.P42A, ZNF517:p.V349A, LRIG1:p.L24V, CCDC102A:p.R96W, OPRD1:p.C27F, SOWAHA:p.R124P, LACTB:p.M5L, TOR3A:p.F13L, ZFPM1:p.E444fs, ZNF787:p.D367 del,LRIG1:p.L26V,IRX3:p.L422P,TRIOBP:p.H1300R,TUBA1C:p.L146F,ZFPM1:p.P445fs,ZFPM1:p.446_44 7LA>P,TPO:p.S398T,USP42:p.R779P,ERCC2:p.D312N,GLTPD2:p.D209E,OTOP1:p.LLW104del,RINL:p.P402 L,AMDHD1:p.S3G,ASPDH:p.Q266R,KCNK17:p.S21G,TMEM247:p.Q128E,MUC5B:p.D682G,OBSCN:p.R4516W,FA M184B:p.R784W,SEMA5B:p.V840D,ZNF598:p.E25G,ADAD2:p.G44E,C1orf106:p.R538C,ZAR1:p.Q42H,PANK2 :p.G126A,PODXL:p.28_30PSP>P,SALL3:p.L593V,THEM4:p.L17R,C2orf81:p.T315P,CLDN23:p.V210M,FAM1 09A:p.GGG156del,FPGS:p.I22V,HHIPL1:p.V692A,MUC5B:p.M2869T,PLEC:p.R1386Q,SYT8:p.R373W,TAF5: p.S130A,TMEM189-UBE2V1:p.N6D,UQCRFS1:p.S6A,B3GNT6:p.L316fs,CCDC105:p.P499T,CLIC6:p.Q298E,IDUA:p.T374P,NOTCH2:p.C19W,RGS9BP:p. A96S,RREB1:p.G783V,SP8:p.G165del,WDR34:p.W60G,C19orf10:p.G12R,CELSR2:p.16_17insP,FAM75C1:p.71_71H>HLVSQRH,GPRIN2:p.R446H,KBTB D13:p.A81V,OGFR:p.S557T,PODXL:p.30_30P>PSP,BHLHE22:p.L62Q,C4orf32:p.G32E,C5orf65:p.Q245R,KNDC1:p.V806D,KRTAP10-6:p.49_49P>PSC CAP,LRP11:p.P92R,MAP1S:p.S411C,NOL9:p.S58A,RASIP1:p.R601C,RGMB:p.S63R,SARM1:p.R23P,TSC22D2:p.A419T,ZNF628:p.T230A,ZNF814:p.A3 37V,AATK:p.A541T,BTBD11:p.G265A,CRIPAK:p.C143R,KCTD3:p.F9V,KRT8:p.S59A,MUC5B:p.S681G,NCOR2:p.1846_1847insSSG,OGFR:p.E556K,APO E:p.C130R,C10orf95:p.A85S,C13orf33:p.R59G,CRIPAK:p.C174R,FAM18B2:p.C51Y,GLI3:p.P998L,GLTSCR2:p.Q389R,HECTD2:p.P19A,IRF2BPL:p. 123_125QQQ>Q,MEX3C:p.179_182AAAA>A,NEFH:p.EE658del,RNF149:p.S9G,RNF222:p.A133T,SEZ6L2:p.R74P,TNIP2:p.R73G,ARRDC4:p.T79A,B3GNT 6:p.P330fs,BAG1:p.G45R,C22orf26:p.P28L,CHDH:p.E40A,COQ2:p.V66L,CTGF:p.H83D,DLEU7:p.A83V,EPPK1:p.D2378H,FAM86C1:p.R30P,FZD1:p.93_94insP,GPRIN2:p.V241M,GPX1:p.11_13AAA>A,HES3:p.P96T,JMJD4:p.A11V,KANK3:p.R359H,LPPR2:p.A18 6S,NEFH:p.665_666insEE,NOM1:p.R24G,RNF39:p.G263C,SCRT1:p.S133A,SNED1:p.L1228P,TTLL11:p.122_123 insKA,ZCCHC3:p.A159del,ZNF219:p.QP233del,ASB16:p.T249A,ASB2:p.H515P,ATP9B:p.S39G,AVL9:p.G7fs,C 17orf96:p.L63V,C19orf29:p.A499V,CRB2:p.T1110M,CRIPAK:p.P173R,CRIPAK:p.I190L,CSGALNACT2:p.L362F ,CTBS:p.LAL31del,CTNNB1:p.S45P,DMRT1:p.S45T,DOK7:p.G461D,FBRSL1:p.A836V,FEZ2:p.P50L,FRG1:p.S1 69N,HSD17B1:p.G313S,IBA57:p.S130R,KIF1A:p.E917D,KRTAP9-1:p.160_160Q>QPSCGSSCCQ,LURAP1L:p.55_56 This includes any combination of mutations selected from the group consisting of insGGG, NMU:p.A19E, NMU:p.A18E, NOXA1:p.D6E, NPTX1:p.G100D, PLIN5:p.R306W, TBP:p.95_96insQ, TMEM200C:p.S498G, TNXB:p.V706fs, VARS:p.P51S, ZC3H12D:p.P405S, and ZZEF1:p.V30A. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0110] 60. (a) The above group of subjects suffers from CESC, (b) At least one of the above tumor-specific mutations is PIK3CA:p.E545K, PIK3CA:p.E542K, MAPK1:p.E322K, EP300:p.D1399N, ERBB2:p.S310F, ERBB3:p.V104M, KRAS:pG 12D,ANKRD12:p.E721Q,ANKRD36:p.M1144T,MICA:p.G318fs,PIK3CA:p.E726K,PTEN:p.R130Q,ABCD1:p.S606P,ACTL7B:p.E211K,ADAM21:p.F129C, ADAMTS12:p.P1053A,AKT1:p.E17K,ANKLE1:p.V643L,ANO3:p.M956I,AOAH:p.R326T,APOD:p.S115L,ASCC1:p.H207Y,ATM:p.S800F,AURKA:p.S387L, BAG5:p.M286I,C12orf43:p.E28Q,C16orf3:p.G65S,C3orf70:p.S6L,C4orf21:p.E800Q,CALB2:p.K60N,CALCB:p.R81T,CCDC152:p.E153Q,CCDC53:p .R58C,CDC27:p.P242S,CFHR5:p.R441H,CLOCK:p.L123fs,CMYA5:p.E2733K,CNTRL:p.P185S,CSHL1:p.R117Q,CSMD3:p.H952Y,CTNNB1:p.D32G,CTSH :p.E254Q,DHPS:p.F49L,DMPK:p.R44H,DNAH14:p.F622fs,DNAH3:p.E3367Q,DNAH8:p.E587D,DNASE1L1:p.D212N,ECE2:p.D254N,FAM71B:p.H445D,F AM73A:p.G23V,FAS:p.E261K,FBXW7:p.R505G,FBXW7:p.R465C,FEZF2:p.E 82K,FKBPL:p.E161Q,FMNL1:p.E927Q,GPATCH3:p.E275Q,GPR142:p.R304T ,GPRIN2:p.T100P,GRAMD2:p.I123M,HERC2:p.S329F,HGF:p.G229A,HIF3A:p.A72T,HIST1H1B:p.K188N,HIST1H2AL:p.R30P,HIST2H2AC:p.R30P,SONG -C:p.N104K,HLA-DPB1:p.G114fs,HRNR:p.G2539S,INVS:p.R799K,JPH3:p.Q433H,JUP:p.S627L,KIAA1211:p.R308fs,KIAA1211:p.E309fs,KLK2:p.E161K,KRAS:p.G13D,KRAS:p.G12V,LIN9:p.E231K,LOC151174:p.P90S,LRRC37A3:p.A406D,LRTM2:p.L176V,MEPE:p.S30T,MUC12:p.R2634C,MUC4: p.S2936L,MYOM2:p.D988N,NFE2L2:p.D29H,NOTCH2:p.R2298W,NPIPL1:p.P250L,NR5A2:p.E80K,NYAP2:p.R197Q,OBSL1:p.E1642K,OR13C2:p.L9V,O SBP:p.Q721H,PAOX:p.H107Y,PDILT:p.E500K,PIAS3:p.D460N,PLEKHO2:p.E351Q,PNRC1:p.R73C,PPP4R1:p.L597F,PREP:p.F469L,PRKDC:p.Q3568E ,PSME3:p.R231W,RANBP6:p.R915W,RCAN2:p.D440N,RNPC3:p.E116fs,SDHAP1:p.H66Y,SDHAP2:p.S37fs,SERPIN3:p.K158N,SERPIN4:p.R98C,SF1 :p.R255W,SGSM1:p.E818K,SIM1:p.V213M,SLC10A4:p.F281L,SLC25A5:p.I79F,SLC35G2:p.K62fs,SLC4A9:p.R617C,SLCO2A1:p.M479I,SND1:p.Q38 E,SPATA17:p.R72K,SRSF12:p.S150C,TADA2B:p.E67K,TCTEX1D2:p.S74L,TEDDM1:p.M166I,TEX15:p.E1652Q,TMC2:p.E92D,TMEM131:p.E1319Q,TNK S2:p.T619fs,TNS1:p.Q659del,TP53:p.E285K,TRAF3:p.S9F,TRIM61:p.K98N,TRPM1:p.M996I,TUFT1:p.L101F,U2AF1:p.S34F,UNC93B1:p.V498M,U SP4:p.L259V,VCAN:p.S1308C,WDR17:p.P278S,ZBED4:p.S385L,ZEB2:p.E 1094K,ZFYVE9:p.M1147I,ZNF16:p.R452W,ZNF677:p.R131T,ANDZSWIM4:p.A protrusion is selected from the group consisting of E407K. Including any combination of natural mutations, A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0111] 61. (a) The above group of subjects is suffering from CRC, (b)Schematic diagrams of 1 and KRAS:p.G12D,KRAS:p.G12V,BRAF:p.V600E,K RAS:p.G13D,TP53:p.R175H,PIK3CA:p.E545K,FBXW7:p.R465H,KRAS:p.A1 46T,PIK3CA:p.H1047R,TP53:p.R248W,CDC27:p.D555E,SMAD4:p.R361H,TP53:p.R273H,KRAS:p.G12C,NRAS:p.Q61K,ERBB2:p.V842I,ERBB3:p.V104 M,FBXW7:p.R465C,PIK3CA:p.R88Q,PIK3CA:p.E542K,TP53:p.R273C,TP53:p.G245S,AXIN2:p.G665fs,C16orf45:p.T106N,C20orf26:p.R1088Q,DNM T1:p.E432K,FBXW7:p.R505C,HLCS:p.E362K,HPSE2:p.K58N,KIF14:p.R598Q,KIF18A:p.R17C,KIF20B:p.E991K,KLHL5:p.R326C,KLK2:p.P57T,KRAS: p.G12A,KRAS:p.G12S,LPHN3:p.R1183Q,LRP6:p.R675Q,MYH8:p.R1048Q,NRAP:p.E327K,NRAS:p.G12C,PIK3CA:p.N345K,POSTN:p.R508C,PPP2R1A:p .R183W,PTEN:p.R130Q,RAF1:p.S257L,SDK1:p.T1181M,SGSM1:p.F1117L,TCF7L2:p.R482fs,TP53:p.R282W,TRIM23:p.R289Q,UGT8:p.E102K,ZNF49 1:p.R343Q,A2M:p.R732Q,AADACL4:p.A266T,ABCA8:p.E1158K,ABCA8:p.R842Q,ABCA8:p.A696T,ABCB8:p.R345H,ACACA:p.R1731C,ACADM:p.F48C,A COT9:p.R50Q,ACPP:p.R105Q,ACTL7B:p.R354H,ACTL9:p.R331H,ACVR1:p.S290L,ADAM30:p.S314Y,ADAM32:p.R559Q,ADAMTS16:p.D817N,ADAMTS4:p.R156W,ADCY5:p.R661H,AGMAT:p.V313M,AGPAT4:p.A212T,AKAP12:p.E1282K,AKAP9:p.L3482I,ALB:p.S294L,ALDH1L1:p.A870T,ALG2:p.S302Y,AMOTL1:p.R676Q,AMPD1:p.K502N,AMPH:p.R292W,ANKRD6:p.R479C,APBA1:p.K730N,APBA1:p.E624K,APC:p.E847fs,APC:p.F1354fs,APC:p.M1413fs,APOB:p.R3136C,APOB:p.A43V,APPL1:p.R668W,AQPEP:p.A309T,ARF4:p.R149H,ARFGEF1:p.D1632N,ARHGAP32:p.E1253K,ARHGAP36:p.R128C,ARHGAP36:p.A147V,ARHGAP5:p.D890fs,ARNTL:p.T395M,ARPP21:p.R338H,ARSG:p.V131I,ASCC3:p.R1197Q,ATP10D:p.R311H,ATP6V0A4:p.R191Q,ATP9B:p.R265Q,AXDND1:p.E930D,AXIN2:p.W663fs,B2M:p.L13fs,B3GALNT1:p.R145Q,BACH1:p.R538Q,BAG5:p.D439N,BBOX1:p.F176V,BCL2L11:p.R91Q,BCL7A:p.T52M,BCLAF1:p.R37fs,BEND5:p.R198C,BICD2:p.R162H,BLVRA:p.S44L,BMP3:p.R344W,BNC2:p.R512W,BRPF1:p.R66C,BRWD3:p.R787C,BTBD7:p.S436L,BUB1B:p.F996L,BZRAP1:p.V1627I,C11orf30:p.R1111C,C14orf101:p.E295K,C14orf102:p.D115N,C14orf1. 05:p.R100I,C15orf2:p.V488I,C15orf33:p.D340N,C16orf87:p.R151I,C1RL:p.L351fs,C22orf40:p.P32fs,C3orf39:p.R333W,C5orf30:p.D4N,C5orf4:p.R114Q,C6orf170:p.K724T,C7orf63:p.A10T,CACHD1:p.S720Y,CACNA1A:p.T665M,CACNA2D3:p.A332T,CACNB2:p.R608H,CACNG3:p.V134I,CACNG3:p.A138V,CACNG5:p.G121R,CADM1:p.S190L,CADPS:p.A1073T,CAPRIN2:p.E13K,CARD11:p.R423Q,CASC1:p.R54Q,CASP14:p.R5W,CBFB:p.E152K,CC2D2A:p.R1284C,CCDC18:p.K615N,CCDC60:p.R230H,CCDC81:p.R259I,CCDC88C:p.P1851fs,CCKBR:p.V236M,CD101:p.D283Y,CD101:p.R594Q,CD180:p.N228T,CDC14B:p.R375C,CDCA7L:p.P405fs,CDH10:p.E349K,CDH12:p.D674N,CDH20:p.A134V,CDH23:p.F177L,CDH2:p.D547Y,CDH9:p.F523L,CDK16:p.R108C,CEACAM5:p.L640I,CEP152:p.E21K,CERS3:p.E95D,CHD4:p.R975H,CHD5:p.A801T,CIZ1:p.V668A,CLEC18A:p.R423H,CLTCL1:p.R481W,CMAS:p.R110Q,CNRIP1:p.R102W,COBLL1:p.K732N,COL14A1:p.R1082I,COL17A1:p.P1004L,COL4A6:p.L550I,COL6A3:p.D2792N,COPB1:p.R425C,CORO2A:p.*526R,COX15:p.L86I,CSMD1:p.S781Y,CTCFL:p.E423K,CTDNEP1:p.E126K,CTTNBP2:p.R164C,CYP4B1:p.E434D,DACH2:p.R539C,DBC1:p.V216I,DBF4B:p.S254Y,DCHS2:p.F2149L,DCLK2:p.S549Y,DDI1:p.R275Q,DENND4A:p.P357H,DENND4C:p.R1081Q,DHTKD1:p.R410Q,DISP1:p.R763C,DKK2:p.R230H,DKK4:p.R203Q,DLC1:p.A350V,DLC1:p.E222D,DMD:p.R3195H,DNAH5:p.R982H,DNAH5:p.R224Q,DNAH9:p.D1547N,DNAJC24:p.E61K,DNM1:p.A251T,DNMT1:p.E1531Q,DNMT3B:p.R92W,DOCK10:p.A1830V,DOCK1:p.E864K,DOCK2:p.G170R,DOCK3:p.R1183C,DOCK5:p.E177K,DOK5:p.R274W,DPP8:p.G165R,DPY19L1:p.F378L,DUOX2:p.F880L,DVL2:p.A601fs,EBAG9:p.E187K,EBF3:p.G255fs,EDNRB:p.L450R,EGR2:p.R390H,EHD3:p.E44K,EIF2C1:p.R139Q,ELF3:p.F305fs,ELMOD2:p.T141M,EMR2:p.S75L,ENAM:p.R373H,ENOX2:p.R356W,ENTPD7:p.E327K,EPG5:p.D369N,EPHB2:p.R392H,ERCC6:p.V780I,ERCC6L:p.R505Q,ERRFI1:p.A421T,ESCO1:p.R300Q,ETV6:p.R369W,F8:p.S2269Y,FAM123B:p.F173fs,FAM135B:p.R884H,FAM169B:p.K165N,FAM170A:p.E56K,FAM171B:p.D459N,FAM181A:p.R109H,FAM5B:p.R402C,FBXO11:p.A432V,FBXW7:p.R689W,FBXW7:p.S582L,FBXW7:p.R14Q,FGF14:p.A236V,FHDC1:p.R254W,FHOD3:p.A225T,FHOD3:p.E813K,FMO3:p.F510L,FNDC1:p.R652H,FOXK1:p.R354W,FOXN3:p.P96fs,FPGT-TNNI3K:p.R455H,. FZD3:p.D367N,GABRA4:p.R460Q,GABRA5:p.S126N,GABRB3:p.D500N,GALNTL5:p.R262I,GJA1:p.R362Q,GLRA3:p.L454I,GLRA3:p.F132L,GOLGA4:p.Q1536H,GP2:p.S41L,GPC6:p.A214T,GPLD1:p.R717Q,GPR125:p.R113Q,GPR156:p.F754L,GPR158:p.D566N,GPR21:p.R216H,GPR61:p.A62T,GPR98:p.R4142W,GPRC5A:p.V30I,GRAP2:p.E69D,GRIA1:p.R218C,GRIA2:p.R845Q,GRM7:p.R679Q,GTF3A:p.K306N,HAO1:p.R172C,HARS2:p.R168H,HBB:p.F42L,HCN4:p.R525H,HDAC5:p.A1044T,HGF:p.S467Y,HIPK4:p.R280H,HLA-DMA:p.E84K,HMG20A:p.E248D,HPS3:p.S468L,HRSP12:p.R120Q,HS3ST1:p.E287K,HTR3B:p.R236C,HTR5A:p.R152C,HTT:p.D1548N,HYDIN:p.R1187C,HYDIN:p.R939Q,HYDIN:p.R451Q,HYOU1:p.R158C,IFT172:p.A944V,IGJ:p.R77Q,IL17RA:p.Q803fs,IL1RAPL2:p.T647M,IL3:p.A90T,IL5RA:p.L47I,INPP5D:p.R523Q,INPP5K:p.R263C,IRAK3:p.R267Q,IREB2:p.R419Q,ITGA4:p.T673M,ITGA4:p.F900L,ITIH5:p.A912T,ITK:p.E196K,JAG1:p.A462T,JAK1:p.V310I,KAL1:p.V303I,KBTBD8:p.V549I,KCNA3:p.A415V,KCND3:p.S438L,KCNMB4:p.F209L,KCTD20:p.L314fs,KDELC1:p.L447I,KIAA0528:p.R181Q,KIAA0556:p.R1082W,KIAA1109:p.S4937Y,KIAA1804:p.V474M,KIAA1804:p.R477W,KIF16B:p.R145Q,KIF26B:p.A1114V,KPNA4:p.R29Q,KRAS:p .K117N,KRAS:p.Q61L,KRAS:p.Q61K,KRT6B:p.L197P,L1CAM:p.T186M,LALBA:p.A41T,LA MA4: p.A558V, LBX1: p. R176W, LPAR4: p. R145Q, LRP1B: p. K2623N, LRP2: p. R3043C, LRP2: p.S737L,LRRC18:p.R218W,LRRC31:p.K23T,LRRC7:p.R1389H,LZTS2:p.P100fs,MACF1:. p.S292L,MACF1:p.F722L,MAEL:p.R345C,MAGEE1:p.V380M,MAGI1:p.R1198C,MAP1B:p.E2046D,MAP2: p.K530N,MAP2K4:p.R287H,MAP3K4:p.R275Q,MAP7D2:p.R487C,MAPK8IP1:p.L217fs,MBOAT2:p.R43Q, MCF2L2:p.R926Q,MECOM:p.R969C,METTL16:p.R200Q,METTL21A:p.R174Q,METTL6:p.F56L,MFF:p.R16 2C,MFSD5:p.R280Q,MIA3:p.Q356H,MMAA:p.R326C,MORC1:p.D113Y,MORC2:p.R740H,MPDZ:p.L804I,M R1:p.S46L,MRPL47:p.L234I,MS4A8B:p.S3L,MSH4:p.K464N,MSH6:p.T1085fs,MSH6:p.R1095H,MUC16 :p.R8606H,MYH13:p.D311N,MYH7:p.R1689C,MYO1D:p.E246K,MYO3A:p.N525H,MYO6:p.D1180N,MYO9A :p.R2179Q,MYO9A:p.R167Q,MYOZ2:p.E251K,MYT1:p.E226K,NAA25:p.S807Y,NCAM1:p.R474W,NCOA4: p.R562Q,NEB:p.D5434N,NEB:p.L1591I,NEB:p.E1214K,NEDD9:p.A798T,NEDD9:p.A316T,NEK1:p.R60 8C,NFASC:p.V256I,NINL:p.R1366C,NLRC4:p.D593N,NLRC4:p.E409K,NLRP4:p.V229I,NLRP5:p.R392H,NME9:p.E75K,NOLC1:p.T428M,NPC1:p.E45 1K,NPSR1:p.R235Q,NRAS:p.Q61L,NRAS:p.G13R,NRAS:p.G12D,NRG2:p.T246M,NTN4:p.E59K,NUB1:p.R373Q,NUDT15:p.S83Y,NUF2:p.S340L,NUP88: p.A302V,ODZ1:p.R2556W,OGDHL:p.A427T,OGFRL1:p.E427K,OLFM4:p.K132N,OPRM1:p.R353H,OR10A3:p.S93Y,OR2M3:p.R235H,OR52W1:p.R133C,O R5AU1:p.R312H,OR5B17:p.R163H,OR8S1:p.A99V,OSTN:p.R115Q,OTOL1:p.V431I,OTUD3:p.R277I,PAN3:p.S580N,PANK3:p.R260I,PAX3:p.T424M,P CBP1:p.L102Q,PCDH10:p.V477M,PCDH15:p.R1552I,PCDHAC2:p.A519T,PCDHAC2:p.E190K,PCDHAC2:p.A266T,PCDHAC2:p.A156V,PCDHAC2:p.E271K ,PCDHAC2:p.A736V,PCDHB5:p.D51Y,PCDHB8:p.D235N,PCDHGC5:p.S289L,PCDHGC5:p.V662M,PCNXL2:p.R135Q,PCOLCE2:p.A348V,PCOLCE2:p.R87H, PDE4B:p.S417L,PGAM1:p.R240H,PHF3:p.R1410I,PIAS2:p.S519L,PIGR:p.A580T,PIK3CA:p.D350G,PIK3CA:p.E545A,PIK3CA:p.E545G,PIK3CA:p. Q546K,PIP4K2C:p.R204H,PKHD1L1:p.F1856L,PLA2G4A:p.E443K,PLCG2:p.E544K,PLCG2:p.D973N,PLEKHA6:p.V328fs,PLEKHG4B:p.E384K,PLK1:p.D233G,PLOD3:p.R297fs,PLSCR3:p.E77K,PLXNC1:p.S462L,PLXNC1:p.R819C,POLA1:p.E603D,POLE:p.S459F,POLE:p.V411L,POLQ:p.R860Q,PPP2R2B:p.P326L,PPP2R5C:p.S259Y,PRAMEF4:p.R248H,PREX1:p.V731I,PRKAA2:p.R407Q,PRKAR2B:p.S309L,PRKCI:p.R480C,PRKRA:p.K122N,PSG8:p.R397C,PSG8:p.R320C,PSMD12:p.R201Q,PTPDC1:p.R430W,PTPN12:p.R765Q,PTPN13:p.S887L,PTPRD:p.L1053I,PTPRU:p.D1434N,PXDN:p.P856fs,PXDNL:p.T1312M,QRSL1:p.S226L,RAB7L1:p.R79W,RALGAPA1:p.R398C,RANBP2:p.R1231C,RBBP7:p.E313K,RBBP7:p.E274K,RBFOX2:p.A340T,RBMXL1:p.R331Q,RHOBTB1:p.T464M,RIMS2:p.R599Q,RIN3:p.S708L,RLBP1:p.D281N,RLBP1:p.A72V,RNASET2:p.A127V,RNF113B:p.A172V,RNF150:p.R236Q,RNF150:p.S208L,RNF43:p.S216L,ROR2:p.D672N,RPL6:p.F193C,RPS6KA5:p.E166K,RSPO2:p.R28C,RUVBL1:p.E431K,RUVBL1:p.R117C,RWDD2B:p.R254H,RXFP3:p.R113C,RYR3:p.R2705Q,SAGE1:p.R229C,SCFD2:p.R545W,SCML4:p.R194Q,SCN10A:p.T1570M,SCN11A:p.A1688T,SCN11A:p.V1289I,SCN11A:p.V566I,SCUBE2:p.V342M,SEMA3A:p.D81N,SEMA4D:p.R252Q,SEPHS1:p.R371Q,SEZ6L:p.S207L,SFPQ:p.R611Q,SFSWAP:p.S617Y,SGCG:p.A220V,SGCZ:p.I41M,SH3T. C2:p.R89C,SIGLEC11:p.S363F,SIPA1L1:p.R1063Q,SIPA1L1:p.S1227Y,SLC12A1:p.S292L,SLC22A15:p.S201L,SLC24A2:p.A134V,SLC25A40:p.R96Q,SLC2A7:p.A65T,SLC30A9:p.R194H,SLC33A1:p.S542L,SLC35F3:p.A280T,SLC39A7:p.R382C,SLC43A1:p.P133L,SLC43A3:p.R216H,SLC44A5:p.R185H,SLC6A2:p.A562T,SLC8A1:p.R431H,SLFN12L:p.F232fs,SLITRK1:p.R52H,SLITRK3:p.S298L,SMAD2:p.R321Q,SMARCA4:p.R381Q,SOCS5:p.S464L,SORBS1:p.V1156M,SORBS1:p.F570L,SORCS2:p.R320W,SOX6:p.R719W,SPATA22:p.S150L,SPEG:p.A944V,SPTB:p.R86C,SPTBN4:p.A1993V,STIM2:p.R572Q,STT3B:p.D583Y,SULT1C4:p.R85Q,SUN3:p.E128K,SUPT6H:p.A957T,SYNE1:p.I1249L,SYNE1:p.R170W,SYNE2:p.K3103N,SYNGR4:p.R169Q,SYT7:p.T349M,TANK:p.S380L,TAS1R2:p.R270C,TAS2R1:p.F183L,TCF7L2:p.R488C,TDRD10:p.S322L,TECTB:p.L29I,TEKT5:p.R401H,TGFBR1:p.S241L,THAP5:p.S287Y,THSD7B:p.R90H,TLL1:p.T153M,TLL2:p.S872L,TM9SF2:p.R91H,TMCC3:p.R110H,TMEM132A:p.R481C,TMEM132D:p.R578W,TMEM55A:p.R189Q,TMEM74:p.R125Q,TMPRSS11A:p.S288L,TNIP2:p.A139T,TOP2B:p.R656H,TOX:p.S354L,TP53:p.G244D,TP53:p.R175C,TPO:p.A826T,TPR:p.S2155L,TPTE2:p.R258Q,TPTE:p.S423L,TRAK1:p.D627N,TRAPPC11:p.R568Q,TRIM23:p.R396Q,TRIM44:p.D331N,TRIO:p.R661W,TRPA1:p.K54N,TRPC5:p.S490L,TRPM6:p.R995H,TRPM7:p.R1862C,TRPM7:p.R843Q,TRPS1:p.R1125W,TRPV5:p.R492H,TRRAP:p.R3515W,TSHZ1:p.R881M,TTC21A:p.S270Y,TTN:p.R22795C,TTN:p.R3193Q,TTN:p.R328H,TUBA3D:p.R243Q,TUFT1:p.A340T,TXNDC15:p.R343Q,UBE2NL:p.R86I,UBIAD1:p.A97T,UGT2A1:p.N97fs,USH2A:p.F2369L,USP11:p.A286T,USP25:p.R1119Q,USP26:p.R861Q,USP29:p.F81L,USP31:p.D391N,USP40:p.S851L,UTP14A:p.V148I,VAV3:p.E685K,VCAN:p.R1125H,VPS13C:p.D1359Y,WBSCR17:p.R228C,WDR3:p.E841K,WDR52:p.A157T,XKR6:p.R268Q,XPOT:p.R541W,YTHDC1:p.R267Q,YTHDC2:p.E634K,ZBBX:p.R596I,ZBTB24:p.L607I,ZC3H13:p.R103Q,ZCWPW2:p.D144N,ZEB2:p.R156H,ZFHX4:p.E237D,ZFP14:p.R386C,ZFP28:p.R525I,ZFP2:p.R150I,ZFP3:p.R273I,ZFP90:p.R330Q,ZHX2:p.V790I,ZIC4:p.S305L,ZIM3:p.D352N,ZKSCAN4:p.R319Q,ZMYM4:p.R1446Q,ZNF117:p.R185I,ZNF167:p.R683I,ZNF180:p.R401I,ZNF19:p.R349I,ZNF205:p.R384C,ZNF236:p.S1480L,ZNF248:p.R568I,ZNF259:p.R174I,ZNF266:p.R512Q,ZNF266:p.R3. 44Q,ZNF280B:p.E363K,ZNF283:p.R392Q,ZNF32:p.S62L,ZNF345:p.R82Q,ZNF345:p.R334I,ZNF350:p.R310Q,ZNF434: p.R306C,ZNF439:p.E239D,ZNF439:p.R262I,ZNF443:p.R301I,ZNF445:p.L682M,ZNF470:p.R641I,ZNF471:p.R282I,ZN F484:p.R138C,ZNF528:p.R279Q,ZNF563:p.K26N,ZNF573:p.R350I,ZNF583:p.R344I,ZNF585A:p.E638K,ZNF585A:p.E 491D,ZNF625:p.R235Q,ZNF652:p.K327N,ZNF677:p.R451I,ZNF678:p.R368I,ZNF699:p.R41I,ZNF70:p.R244I,ZNF770: This includes any combination of mutations selected from the group consisting of p.S441P, ZNF774:p.R423Q, ZNF782:p.K247T, ZNF7:p.R337I, and ZNF831:p.E949D. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0112] 62. (a) The above group of subjects suffers from DLBCL, (b) At least one of the above tumor-specific mutations is EZH2:p.Y641F,MYD88:p.L273P,BCL2:p.G33R,CARD11:p.E626K,ADCY2:p.A87V,BCL2:p.N172S,BCL2:p.H20Q,BRAF:p.K601E,BTG1:p.L31F,CACNA1E:p.R1458C,CARD11:p.E93D,CD79B:p.Y197D,CD79B:p.Y197H,CREBBP:p.R1446H,GRID1:p.E622K,HIST1H This includes any combination of mutations selected from the group consisting of 1C:p.A65V, HIST1H1E:p.G133A, HIST1H3B:p.A48S, KRAS:p.G13D, MYD88:p.S251N, PABPC1:p.R94C, PIM1:p.L164F, PIM1:p.L184F, POU2F2:p.T239A, POU2F2:p.T239S, RELN:p.R2971Q, SLC25A48:p.A67T, STAT6:p.D468H, TNF:p.L47F, and TRAF7:p.R11H. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0113] 63.(a) The above group of subjects is suffering from KICH, (b) At least one of the above tumor-specific mutations is: ACR:p.W279C,AGRN:p.1284_1285VT>A,C7orf25:p.R384fs,CAMSAP1:p.T466fs,CBWD6:p.E102fs,DOCK8:p.L1111fs,EBPL:p.Q196P,EBPL:p.L189V,GFM1:p.A17fs,GOLGA6L6:p.D570E,ITGA5:p.A48D,LUZP2:p.S154fs,MTM This includes any combination of mutations selected from the group consisting of R9:p.K193fs, MUC16:p.P10452fs, MUC4:p.S2832P, ODF2L:p.K407fs, RHBDD3:p.G34fs, RILPL1:p.S358R, TAS2R30:p.L236fs, TRRAP:p.A973S, UBR5:p.K2120fs, URGCP:p.G639fs, ZNF98:p.A222T, and ZSWIM6:p.Q610fs. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0114] 64. (a) The above group of subjects is afflicted with KIRP, (b) At least one of the above tumor-specific mutations is FAM18B2:p.C51Y, ZNF598:p.E25G, NEFH:p.E645K, EEF1B2:p.S43G, NEFH:p.AKSPEKEE652del, OBP2B:p.K61N, SKI:p.A62G, C14orf126:p.R6W, KRT8:p.S59A, ACSBG2:p.I250M, ASIC2:p.R46L, CSGALNACT2:p.L362 F,FRG1B:p.A50P,IDUA:p.H33Q,KRTAP4-5:p.S74C,SCAF11:p.E926fs,SYN2:p.A34del,ZNF814:p.R322K,BMS1:p.E878D,JMY:p.P822T,KIF1A:p.E91 7D,KRTAP4-7:p.S57P,LAMA5:p.L2223R,LRP1:p.P1058T,MED16:p.H449Q,MUC2:p.T1488P,MUC5B:p.D682G,NACA2:p.R75K,NEFH:p.665_666insEE,OR 2L8:p.S201fs,RGPD5:p.P1760A,RRN3:p.P11S,RRN3:p.R9C,STAG3L2:p.L81fs,ZNF814:p.G320E,ACP6:p.V29G,AHNAK2:p.S2166F,AHNAK2:p.P1215 S,AP1G1:p.I782fs,AQP2:p.N68T,BAIAP2L2:p.V396M,BMP6:p.Q118L,BST1:p.G36A,CDR1:p.V31A,CLDN7:p.S172A,CLIP1:p.S1018fs,COL18A1:p.G8 84fs,CROCC:p.A355P,CTAGE15P:p.A364V,CUBN:p.I2816M,DMRT2:p.T106S,DPY19L1:p.V249L,DSPP:p.D1047N,EBPL:p.L189V,EIF4G1:p.E465del, EXOSC2:p.R11P,FAM216A:p.P36S,FCGR2A:p.V222G,FMOD:p.S331R,FOLR2:p.Q112R,FRG1B:p.L20P,GAGE2B:p.9_10insY,GDPD5:p.G593fs,GIMAP8:p .A544S,GLUD2:p.R300G,GLUD2:p.S496R,GPR135:p.Q5P,HOXD8:p.Q67H,IER5:p.R194G,IL25:p.C168fs,JSRP1:p.V92A,KRAS:p.G12D,KRTAP1-1:p. Y86C,KRTAP4-11:p.L161V,LTBP1:p.L163P,MAML2:p.Q591K,MAPK7:p.A501D,MEF2A:p.P99S,MET:p.H1094Y,MET:p.M1250T,MST1:p.N435fs,MUC2:p.T1582R,MUC2:p.T1722I,MUC4:p.A4222T,MUC4:p.T2335M,MUC4:p.P1138L,MUC5B :p.S1098A,MUC5B:p.S3431N,MYH7:p.A1487T,NBPF10:p.R39fs,NBPF10:p.Y638S, NEFH:p.654_654S>SPEKAKS,PARG:p.A584T,PBX2:p.Y262F,PIP4K2A:p.R219K,RLI M:p.S471P,RUNX2:p.Q71E,SGK223:p.R63S,SMARCB1:p.L365fs,SRCAP:p.Q1875fs This includes any combination of mutations selected from the group consisting of TBC1D2B:p.R920Q, TCF7L2:p.R482fs, TMEM131:p.K640fs, TMEM60:p.K77fs, TPPP:p.R30K, TRPV3:p.A218E, TTBK2:p.C83W, UBXN11:p.S510G, UGT1A1:p.T4A, UTS2R:p.A289E, YBX1:p.P250L, ZNF514:p.V81G, ZNF516:p.A256D, ZNF681:p.K405Q, ZNF814:p.D404E, ZNF814:p.P323H, and ZXDB:p.G206R. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0115] 65. (a) The above group of subjects is suffering from LIHC, (b) At least one of the above tumor-specific mutations is TP53:p.R249S, CTNNB1:p.D32V, CTNNB1:p.D32G, CTNNB1:p.S33P, CTNNB1:p.K335I, CTNNB1:p.H36P, EEF1A1:p.T432L, GNAS:p.R844C, OR2T4:p.V137L, TP53:p.H1 93R,ATXN1:p.Q217H,CSMD3:p.F2383fs,CTNNB1:p.D32N,CTNNB1:p.S33C,CTNNB1:p.G34V,CTNN B1:p.S45P,CTNNB1:p.N387K,DHRS4:p.I218T,DNM2:p.E378D,F5:p.Q426L,GALNTL5:p.A45T,GP This includes any combination of mutations selected from the group consisting of X1:p.P77R, GRM8:p.R852C, IDH1:p.R132C, KIF26B:p.A2033T, KRT8:p.S59A, LOC100132247:p.T532P, NEB:p.D3854H, PIK3CA:p.H1047R, SOLH:p.R714H, TP53:p.R158H, TP53:p.V157F, and ZNF638:p.D400N. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0116] 66.(a) The above group of subjects is suffering from MM, (b) At least one of the above tumor-specific mutations is NRAS:p.Q61R,KRAS:p.Q61H,KRAS:p.G13D,NRAS:p.Q61K,BRAF:p.V600E,NRAS:p.Q61H,NRAS:p.G13R,ZNF717:p.W315C,ATP13A4:p.V431G,DNAJC12:p.R135K,IRF4:p.K123R,KRAS:p.A146T,KRA S:p.Q61R,KRAS:p.G12A,KRAS:p.G12D,ZNF717:p.N594I,ACTG1:p.A22P,ARL6IP1:p.M75L,BEND2:p.E630K,BR AF:p.G469A,CDHR1:p.R218G,DIS3:p.R780K,DMXL2:p.D2412E,DNAJC10:p.I80K,EGR1:p.Q9H,FGFR3:p.*807S, IDH1:p.R132C,IL6ST:p.P216H,INTS12:p.M1V,KRAS:p.K117N,KRAS:p.A59G,KRAS:p.G12R,MAX:p.R36W,MLL5 :p.G492E,NBPF1:p.E810K,NRAS:p.Q61L,NRAS:p.G12D,ODF2L:p.E294K,PADI2:p.T114P,PNLIP:p.T37M,PRDM1 This includes any combination of mutations selected from the group consisting of :p.S588C,PTPN11:p.E76K,PTPN14:p.E286K,RBM6:p.V675G,SCN10A:p.R1142H,SRGAP1:p.T61M,SUSD1:p.T168P,TAS2R16:p.V231I,TINAG:p.E403K,TRIP12:p.L1775P, and ZNF717:p.C844S. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0117] 67.(a) The above group of subjects suffers from PRAD, (b)Schematic diagrams of 1 and HSD17B7P2:p.N175S,RGPD5:p .P1760A,FRG1B:p.L52S,EEF1B2:p.S43G,FRG1B:p.I10T,FR G1B:p.A53T,LRRC37A2:p.T102S,NBPF10:p.E3455K,PTH2:p.L22V,CYP2D7P1:p.S32A,FAM47C:p.N648D,MAP3K9:p.E38d el,MUC4:p.H4205Q,CHEK2:p.K373E,FRG1B:p.A11T,FRG1B:p.A50P,HLA-J:p.R124W,KRTAP1-5:p.I88T,KRTAP4-9:p.D1 8V,NPIP:p.A271V,PDGFRA:p.R483fs,ZNF780A:p.Q600H,ZNF845:p.R925H,ZNF91:p.R333H,ARFGAP3:p.N299fs,BTN2A3 P:p.P3S,FNBP4:p.TT58part,HLA-A:p.Q78R,LOC554223:p.RAPWMEQ147part,PODXL:p.28_30PSP>P,POLI:p.D17part,SPOP :p.F133L,SYN2:p.A34del,TMEM52:p.23_26LLPL>L,UBC:p.L149R,ZNF208:p.I647S,ZNF799:p.E589G,ZNF814:p.D404E ,ASTN2:p.L221Part,B4GALNT1:p.G88fs,C16orf74:p.S21Part,CCDC15:p.H458P,CD209:p.R129W,CNTNAP1:p.S1029I,DB R1:p.541_542DD>D,FAM22F:p.S691del,FRG1B:p.D32V,FRG1B:p.I34T,FRG1B:p.N55D,FRG1B:p.I59V,FRG1B:p.S71N,K IF25:p.W3R,KRTAP4-11:p.L161V,KRTAP4-11:p.M93V,KRTAP4-11:p.R51K,KRTAP4-6:p.S153Y,LILRB5:p.S598P,LMOD2:p.E124del,LOC645752:p. L40P,LRP1:p.P1058T,LRRIQ3:p.K244fs,LURAP1L:p.55_56insGGG,MLLT10:p.V463E,MYOCD:p.Q310del,NBPF10:p.N1369D,OTUD4:p.T909I,PARG:p .A584T,PEX1:p.I370fs,POTEC:p.K507E,POTEC:p.R477Q,POU4F2:p.68_69insG,PRG4:p.T417P,SDHAP2:p.R31C,SPOP:p.F133C,SPOP:p.W131G,TI MD4:p.T152del,TMEM121:p.P299del,TP53:p.G245S,UBC:p.R73L,UBC:p.I191T,WASH3P:p.G175S,ZMIZ1:p.D1048fs,ZNF709:p.T413I,ACADS:p.R3 30H,ADAMTS7:p.K1357fs,AFF2:p.R597H,AGAP6:p.S127I,AK302238:p.A44T,AK302879:p.Q191R,ALDH1A2:p.R85C,ANAPC1:p.T537A,ANKRD36C:p. H438R,AP4B1:p.R276W,ARFGAP2:p.S38N,BBS9:p.F268fs,BC139719:p.L133R,BRAF:p.G469A,C22orf43:p.D171del,CANT1:p.K131R,CHD3:p.E35de l,CLEC4A:p.R209H,CNOT3:p.E20K,CNPY3:p.17_18LL>L,CNTNAP3B:p.S317T,CNTNAP3B:p.M1247I,CTNNB1:p.T41A,DDX10:p.D788del,DLC1:p.S741 T,DPY19L2:p.M210V,EDC4:p.S617del,EFCAB6:p.R379K,ERC2:p.927_928HH>H,FAM111B:p.S269fs,FEM1A:p.L620M,FHOD3:p.A632fs,FLJ43860:p.L850fs,FMN2:p.G59part,FNBP4:p.914_915PP>P,FRG1:p.E86part,FRG1B:p.K13N,FRG1B:p.P42Q,GABRB1:p.R416C,GABRR2:p.A368V,GAGE2B:p.9_10i nsY,GOLGA8DP:p.N84H,GOT2:p.R355W,GPATCH4:p.K210fs,HDGFL1:p.188_189insA,HLA-DQB2:p.G250S,HLA-DQB2:p.R247H,IDH1:p.R132H,IL27:p. E176part,IRF2BPL:p.123_125QQQ>Q,KANK3:p.DGDS489part,KIAA1462:p.858_859SS>S,KRTAP4-11:p.S48R,KRTAP4-7:p.S57P,KRTAP4-8:p.C95S,LPH N3:p.R826H,LRP10:p.L11del,LRP5:p.S1609P,LRRC16B:p.R787W,MAS1L:p.R324G,MECOM:p.R915Q,MED12:p.L1224F,MED12L:p.Q2115del,MESP2:p. GQGQGQGQ195part,MGAT4C:p.T345M,MLEC:p.E238part,MSLNL:p.T68P,MUC7:p.S173P,MYC:p.Q37part,NBPF10:p.N440D,NLRP6:p.E611part,NOX3:p.C40 4fs,OR1M1:p.V69I,OR7E24:p.L7fs,OTUD4:p.A153del,PANK2:p.T417fs,PCLO:p.S496P,PCNT:p.S162G,PCSK9:p.23_24insL,PHOSPHO1:p.S32del,P OU4F1:p.H108part,PRAMEF8:p.R319H,PRDM7:p.M387L,PRG4:p.T597P,PTPRD:p.R1323C,PTPRF:p.R1174Q,ROBO3:p.RS1367part,ROCK1:p.T518S,RPTN :p.G296S,RTL1:p.152_152E>EE,SIRPA:p.V233I,SLC2A6:p.A230D,SLC8A2:p.E710part,SMG7:p.E846fs,SNAPC4:p.S542part,SP8:p.G165part,SPOP:p.F133I, SPOP. :p.F133V,SPOP:p.F102C,SPOP:p.F102V,SRSF11:p.G17fs,SRSF4:p.K396del,SSPO:p.S4198fs,STAG3L2:p.L81fs,STK 19:p.R18fs,TBC1D2B:p.R920Q,TBC1D9:p.P1233T,TCHH:p.P1158R,TCOF1:p.K1366del,TNRC18:p.2664_2665SS>S,TP5 This includes any combination of mutations selected from the group consisting of 3:p.R248Q,TP53:p.R175H,TP53:p.C141G,TSPAN4:p.L92V,UBXN11:p.GPGPGPSP504del,UTP3:p.E81del,WASH3P:p.L187V,ZAN:p.P717L,ZAN:p.L878P,ZFP90:p.R591fs,ZNF761:p.H373R, and ZNF91:p.H305R. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0118] 68. (a) The above group of subjects is suffering from STAD, (b)Schematic diagrams of 1 and RNF43:p.G659fs,BZRAP1:p.P1416fs,XYLT2:1. p.Y526fs,LARP4B:p.T163fs,PGM5:p.I98V,ZBTB20:p.P692fs,ARID1A:p. G1848fs,FHOD3:p.P334fs,KIAA0182:p.T120fs,ATP6V1B1:p.Y383fs,PIK3CA:p.H1047R,FRMD4A:p.P1005fs,PIK3CA:p.E545K,CDC14A:p.N123fs,K RAS:p.G13D,MLL2:p.T172fs,BCORL1:p.S1679fs,PLEKHA6:p.V328fs,C9orf131:p.P342fs,CD4:p.Q164fs,FBXW7:p.R465C,GNG12:p.T68fs,IRS4:p .G591fs,JARID2:p.V422fs,KIAA0195:p.I902fs,MBD6:p.P732fs,MVK:p.P138fs,PAMR1:p.G101fs,WNT16:p.W165fs,ZNF43:p.N251fs,ABCA6:p.L30 6fs,ADAM28:p.K73fs,AOC3:p.L79fs,ATP2A1:p.R819fs,B2M:p.L13fs,C6orf89:p.P58fs,CNTLN:p.K1305fs,CR2:p.V206fs,DYRK4:p.K468fs,ERBB 3:p.V104M,GLI1:p.W272fs,KRAS:p.G12D,MLL2:p.T172fs,MSH6:p.T1085 fs,NLK:p.C190fs,OR5M3:p.T89fs,PAX6:p.P375fs,PTEN:p.L265fs,RABGA P1:p.K928fs,RAD51AP2:p.T316fs,SVIL:p.G1862fs,TP53:p.R273H,WNK4:p.G606fs,ARID1A:p.P2139fs,AXIN2:p.G665fs,C13orf33:p.R67fs,C1Q TNF5:p.P308fs,CELSR1:p.G614fs,CRYGD:p.G159fs,DCHS1:p.R235fs,DDC:p.I433fs,EDNRB:p.Y383fs,EPHA2:p.P460fs,FOXN3:p.P96fs,HDAC4:p.P901fs,INF2:p.S527fs,KIRREL2:p.V649fs,KLF3:p.I104fs,KLHL14:p.P231fs,MAP7D3:p.Q308fs,OTX2:p.R44fs,PAFAH1B1:p.K302fs,PLAGL2:p.P10fs,POLM:p.P97fs,PRPF40B:p.I31fs,RALGAPB:p.T379fs,SBNO1:p.N1139fs,SERPINI1:p.L81fs,SH3KBP1:p.L574fs,SLC12A7:p.H686fs,SLC27A3:p.P643fs,TBX4:p.S370fs,TP53:p.R273C,TP53:p.R175H,TRAM1L1:p.R345fs,WBP1:p.P138fs,ABCC4:p.L883fs,AKAP13:p.K2785fs,ALDH3A1:p.P562fs,ALPK2:p.L356fs,ARFGEF1:p.P1552fs,ARID1A:p.G1848fs,AVPR1A:p.F351f. s,BAX:p.M38fs,C14orf43:p.P313fs,C1QTNF5:p.G194fs,C7orf50:p.L179fs,CDC25C:p.K322fs,CETN3:p.K63fs,CHD3:p.P597fs,CTCF:p.K202fs,CTSC:p.F105fs,DDX17:p.G163fs,DLGAP3:p.G377fs,EBF3:p.G255fs,FHDC1:p.F100fs,FILIP1L:p.K749fs,FLNB:p.W529fs,GBP7:p.G431fs,GCC2:p.E700fs,GPR161:p.G517fs,IWS1:p.S802fs,KIAA0240:p.K895fs,KIAA1967:p.P415fs,LRRC43:p.D558fs,MACF1:p.R707fs,MBD6:p.G780fs,MLL3:p.F4496fs,MPRIP:p.A351fs,MUC6:p.2129_2130SS>S,NOX5:p.P467fs,OPTN:p.P24fs,OR4K5:p.F177fs,PIK3CA:p.N345K,PIK3CA:p.E542K,PLXNA1:p.P1016fs,PNPLA7:p.P1199fs,PODN:p.I301fs,PPP2R3B:p.T389fs,PRSS36:p.L680fs,RGL2:p.G203fs,RHOQ:p.V190fs,RNF111:p.R771fs,RTN2:p.P313fs,SALL4:p.V995fs,SBF1:p.P1076fs,SETDB2:p.R715fs,SNAPC2:p.T292fs,SPG20:p.F232fs,SRCAP:p.P1876fs,STAT2:p.P489fs,TCHP:p.E172fs,TP53:p.R282W,TP53:p.R248Q,USP21:p.K474fs,WDR7:p.G262fs,ZBTB7C:p.E157fs,ZFC3H1:p.K385fs,ZNF124:p.T339fs,ZNF626:p.K115fs,ADNP2:p.S322fs,AGAP1:p.G127fs,ALDH2:p.L286fs,ARHGAP5:p.D890fs,ARHGEF17:p.A615fs,ARID1A:p.Y1324fs,ART1:p.I243fs,ASCL4:p.D35fs,ATXN2L:p.G998fs,B3GNT5:p.F30fs,BCKDHA:p.H37fs,BCL9L:p.P1127fs,BEND3:p.D265fs,BNC2:p.S575R,BRD3:p.P24fs,C12orf51:p.P4235fs,C1R:p.P216fs,C7orf49:p.G130fs,CA2:p.I145fs,CABP5:p.R145fs,CASD1:p.F781fs,CASP8:p.R471fs,CCDC153:p.P200fs,CD93:p.D280fs,CROT:p.L32fs,CSF3R:p.P468fs,CTCF:p.K202fs,ERBB2:p.S310F,FAM46D:p.S69R,FBN3:p.G601fs,FBXO21:p.F144fs,GAS6:p.G150fs,GLYR1:p.G380fs,GXYLT1:p.L223fs,HAUS6:p.S530fs,IGF2R:p.T1314fs,ITGB1:p.L378I,KDM3B:p.P1316fs,KIF13A:p.K1115fs,KLF3:p.S224fs,LARP1:p.A223fs,LRP1:p.G1488fs,LRP1:p.G1488fs,MAGEE2:p.Q45fs,MAMSTR:p.P162fs,MAPK15:p.Q511fs,MLL2:p.P647fs,MOCS2:p.P22fs,MTG1:p.L105fs,MTG1:p.H327fs,MTIF2:p.N109fs,NID2:p.R1035fs,PAX2:p.P395fs,PCCA:p.R230H,PDZD2:p.R101fs,PFKP:p.M593fs,PIK3CA:p.R88Q,PLA2G1B:p.L53fs,PLAU:p.R201fs,PMEPA1:p.P208fs,POP1:p.K750fs,PTCH1:p.P1307fs,PTPRT:p.P1075fs,RDBP:p.P6fs,RNMT:p.K392fs,ROBO2:p.P1080fs,RUNDC3B:p.L6fs,SDAD1:p.K275fs,SLC10A6:p.G109fs,SNAPC1:p.D211fs,SPATA5L1:p.C685fs,SPTA1:p.K1732T,STAT5B:p.P367fs,SYT4:p.M1fs,TAF1L:p.K851fs,TAP2:. p.L75fs,TBL1XR1:p.N126fs,THEMIS:p.K406fs,TMEM79:p.P161fs,TP53:p.C176F,TP53BP2:p.K69fs,TP53RK:p.L174fs,UBQLN2:p.A523fs,UHRF1BP1:p.I1330fs,VPRBP:p.K939fs,VPS13B:p.T56fs,WASF3:p.P305fs,YLPM1:p.E1178fs,ZC3H13:p.K1006fs,ZC3H18:p.P825fs,ZC3H4:p.E779Q,ZNF48:p.P247fs,ZNF608:p.A465fs,ZNF878:p.S238fs,ZSCAN18:p.P225fs,ABCB1:p.R527fs,ABCB6:p.G318fs,ACACB:p.G255fs,ACP1:p.Q123fs,ACTL6A:p.L88fs,ADAMTSL4:p.G778fs,AGBL5:p.I420fs,AHI1:p.K303fs,AKAP9:p.M3743fs,AKD1:p.R1209fs,ANKRD40:p.D99E,ARHGEF5:p.S1512fs,ARID1A:p.K1071fs,ARID3A:p.S557G,ARPP21:p.I130fs,ASPN:p.F67fs,ASXL3:p.E873fs,ATP6V1C2:p.R312fs,BEST3:p.P444fs,BRAF:p.P403fs,BRMS1:p.G107fs,BTBD11:p.T451fs,BTBD11:p.A561V,C11orf9:p.S261fs,C14orf102:p.R90fs,C14orf43:p.Q36fs,C15orf52:p.G98fs,C19orf21:p.R262C,C19orf70:p.P50fs,C20orf160:p.P46fs,C3:p.P890fs,CADPS2:p.N468fs,CASC3:p.S232F,CASC3:p.P603L,CASC3:p.P645L,CASC3:p.S658L,CASKIN2:p.P727fs,CBLL1:p.E138fs,CBLN3:p.P69fs,CCDC108:p.P1164fs,CCDC148:p.K420fs,CCDC153:p.P200fs,CCDC169-SOHLH2:p.K162R,CCDC88A:p.K677fs, CD1E:p.F85V, CD3EAP:p.K218fs,CDH11:p.K357T,CDH1:p.D254Y,CDH23:p.V403I,CFI:p.K37 fs,CHPF2:p.D645fs,CIC:p.R507fs,CIC:p.A1114fs,CIC:p.A1114fs,CLSTN1:p.T615M,CNBD1:p.L39 6P,CNGA4:p.K510T,CNOT6:p.S248fs,CNTROB:p.R920fs,COL9A1:p.P283fs,CPAMD8:p.P784fs,CR1L: p.L79fs,CRB1:p.F630V,CSMD1:p.L3410V,CTNNA3:p.K856fs,CTNND1:p.I447fs,CTSD:p.P89fs,CUX1: p.A439fs,CYP7B1:p.K332T,DAB2IP:p.D994fs,DNAH11:p.T871fs,DNAH8:p.K1688fs,DNAJC1:p.K193 fs,DNM2:p.P791fs,DSTN:p.F101fs,DISH1B:p.Q545fs,EAF2:p.V109fs,EDNRB:p.A104V,EEA1:p.N570 fs,EFHA1:p.F290fs,EGR1:p.P332fs,EIF4G3:p.K563fs,ELK3:p.S173fs,ENTPD2:p.G204fs,EOMES:p .G332fs,EPHA10:p.P868fs,EPHB6:p.G54fs,EPHX1:p.P132fs,EPPK1:p.G2015fs,ERBB4:p.M1fs,ESF. 1:p.T99fs,EXOSC8:p.L160fs,FAM113B:p.R51fs,FAM116A:p.L441fs,FAM135B:pS 645R,FAM151A:p.P117fs,FAM193A:p.D428fs,FAM193A:p.D428fs,FAM214B:p.A42f s,FAM40B:p.R740C,FAM70B:p.S19L,FASTKD1:p.K3fs,FBXW7:p.R479Q,FBXW9:p.G2 98fs,FER:p.L474fs,FERMT2:p.K152fs,FGGY:p.G138fs,FIGNL1:p.K309fs,FLG:p. K159fs,FLNB:p.W529fs,FOLH1:p.S501fs,FYB:p.G324fs,GABRD:p.Q412fs,GALNTL1:p.W317fs,GANAB:p.L23fs,GCDH:p.L389fs,GIMAP7:p.V276fs,GIPC3:p.G227fs,GLI3:p.P1033fs,GLIPR1L2:p.G92fs,GNPNAT1:p.F54fs,GON4L:p.M134fs,GPATCH4:p.K210fs,GRK4:p.K22fs,GTF3C1:p.S767fs,GTF3C4:p.E562fs,H2AFY2:p.K144fs,HCFC1R1:p.P83fs,HCRTR2:p.S9fs,HCRTR2:p.S9fs,HDLBP:p.G747fs,HECA:p.R333fs,HIVEP3:p.H554fs,HIVEP3:p.P534fs,HLA-C:p.P209fs,HOOK1:p.L361fs,HOXD8:p.P122fs,HTT:p.G697fs,IBTK:p.K1213fs,IDE:p.K37fs,IFT172:p.A837T,INPPL1:p.A974fs,INPPL1:p.P1154fs,INSM2:p.T533fs,INTS12:p.L14fs,INVS:p.R815fs,IPO11:p.S844fs,IRX6:p.A425V,ISG20L2:p.P288fs,ITGB8:p.A7fs,JARID2:p.G394fs,JHDM1D:p.R97fs,KBTBD6:p.G442fs,KCNC1:p.K455fs,KCNH2:p.G149A,KCNJ10:p.P102fs,KCNMB2:p.N151K,KCTD21:p.T6M,KIAA0586:p.A1592fs,KIAA1009:p.F406fs,KIAA1109:p.E1588fs,KIAA2026:p.K690fs,KIF26B:p.S1065fs,KIF6:p.L204fs,KIRREL:p.P335fs,KLC2:p.T568fs,KRAS:p.Q61H,KRAS:p.G12S,MAN1C1:p.G431fs,MAP1A:p.P2063fs,MAP2:p.K1472fs,MAP3K12:p.R449del,MAP7D1:p.A80fs,MGST2:p.K102fs,MKI67:p.T1664fs,MKL1:p.P307fs,MLL2:p.P2354fs,MLL2:p.L656fs,MLL2:p.P647fs,MLL2:p.L1877fs,MMP3:p.I64fs,MPDZ:p.K1582fs,MTUS2:p.R1005W,MUC16:p.A6156T,MYB:p.R481fs,MYEOV:p.L269fs,MYH11:p.K1263del,MYO18A:p.P209fs,MYO7A:p.I539fs,MYOCD:p.G226fs,NAA16:p.H514fs,NBEA:p.V2247fs,NCAPD3:p.Q909fs,NCAPH:p.T466fs,NCOR2:p.P1308fs,NEFM:p.A213V,NEK8:p.V690fs,NF1:p.T676fs,NHLRC1:p.F204fs,NKD1:p.P286fs,NPR3:p.Y138H,NT5M:p.P206fs,NUFIP2:p.R224fs,NUP210:p.L135fs,NYNRIN:p.G113fs,OBSCN:p.G997fs,OGDH:p.Y948fs,OR4C16:p.S135R,OR51A7:p.L124R,OR7C1:p.C179fs,OSBP2:p.H627fs,OTOF:p.E1304K,P2RX1:p.R20fs,PALB2:p.M296fs,PALB2:p.N280fs,PANK1:p.K400fs,PAPD4:p.C225fs,PAPPA2:p.I1683fs,PARP15:p.K461fs,PARP4:p.K847fs,PCDH10:p.N118fs,PCDH10:p.P225fs,PCGF3:p.H63fs,PELI2:p.G197fs,PHACTR1:p.V251fs,PHACTR2:p.S237fs,PHACTR4:p.S354fs,PHKB:p.K642fs,PIAS3:p.H116fs,PIGO:p.P787fs,PIGT:p.A346fs,PIK3R3:p.M341fs,PITPNM1:p.P295fs,PKN2:p.K76fs,PLA2G15:p.W230fs,PLAG1:p.K184fs,PLEKHO1:p.T254fs,PLOD3:p.R297fs,PLOD3:p.P296fs,PLXNA2:p.P464fs,POLQ:p.L143. 0fs,PPARGC1B:p.P135fs,PPL:p.P454fs,PPM1H:p.P226fs,PPP1R12C:p.P372fs,PREX2:p.R562fs,PRICKLE4:p.Q109fs,PRKAR1B:p.P87fs,PRKCG:p.R345C,PRMT8:p.S28fs,PROX1:p.F592fs,PRRG3:p.R163fs,PSD2:p.G256fs,PTCHD3:p.F588fs,PTPN4:p.N319fs,PTPRC:p.Q895H,PWWP2B:p.S84fs,PYGO2:p.Q150fs,RABGAP1:p.K928fs,RB1CC1:p.N1171fs,RBM6:p.R96fs,RHOA:p.Y42C,RIMS1:p.R71G,RIMS2:p.V401fs,RING1:p.G171fs,RINT1:p.L107fs,RNF43:p.P116fs,ROBO2:p.K1293fs,RPS6KA6:p.K109fs,RRS1:p.N45fs,RSF1:p.K386fs,RUSC2:p.P486fs,RXFP3:p.A60V,SAFB:p.W798fs,SCARF1:p.R614Q,SCLT1:p.K109fs,SERPINB12:p.Q168fs,SGK3:p.L61fs,SGOL2:p.E407fs,SIGLEC1:p.P318fs,SIK1:p.Q678fs,SLC16A6:p.G98fs,SLC25A17:p.F28fs,SLC26A7:p.I629fs,SLC32A1:p.V494I,SLC4A3:p.L1061fs,SLC7A10:p.P157fs,SLC9A2:p.T746fs,SLITRK1:p.K45fs,SND1:p.H721fs,SOAT1:p.F64fs,SORBS2:p.E1158fs,SOX7:p.L309fs,SPAG17:p.Q1264fs,SPTY2D1:p.P485fs,SRCIN1:p.P865fs,SREBF2:p.H763fs,SRRT:p.G102fs,STAB1:p.P1120fs,STRADA:p.R333fs,STX2:p.K252fs,SV2A:p.E138fs,SYCP2:p.M176fs,SYNJ2:p.P1111fs,TAS2R10:p.L196fs,TBC1D22B:p.A175fs,TEAD2:p.P298fs,TFE3:p.G482fs,TGM6:p.T358fs,TIMM44:p.K83fs,TIMP3:p.A199fs,TLR4:p.L498V,TMEM132D:p.P206fs,TMEM41A:p.F 156fs,TMEM41B:p.F230fs,TMTC4:p.R611C,TNK2:p.P632fs,TOPBP1:p.I1381fs,TP53:p.E286K,TP53:p.P152fs,TRIP11:p.K541fs,TRPA1:p.T67 3fs,TRPM8:p.H765fs,TTF1:p.K336fs,TTI1:p.R707H,TTN:p.E15192D,U2AF2:p.L175fs,UBC:p.G684fs,UBR4:p.P2802fs,UPF2:p.E1033D,UPK2: p.P49fs,USP13:p.I116fs,USP15:p.K782fs,VASH1:p.G3fs,VEZF1:p.355_356insN,VPS13A:p.F2883fs,WAPAL:p.R522fs,WDFY3:p.L1842fs,WDR 59:p.N160fs,WDR5:p.N214fs,WDR60:p.Q412fs,WDTC1:p.M287fs,WHSC1L1:p.K418fs,WNT1:p.W167fs,XIRP2:p.E1007D,YBX2:p.P226fs,YIF1A: p.R131fs,ZBBX:p.E151fs,ZBTB40:p.L262fs,ZBTB7C:p.G342fs,ZBTB7C:p.D154fs,ZC3H18:p.T701fs,ZDHHC5:p.E651fs,ZDHHC7:p.P316fs,Z FHX3:p.R1893fs,ZFHX3:p.E763fs,ZFHX4:p.L408fs,ZHX3:p.N249K,ZIM 3:p.I384fs,ZKSCAN5:p.D13fs,ZMYM4:p.K345fs,ZNF236:p.T1410M,ZNF 23:p.F122fs,ZNF334:p.K426fs,ZNF358:p.T130fs,ZNF701:p.L296fs,Z NF711:p.L737fs, and ZNF831:p.A49f This is a great way to get the best results. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0119] 69. (a) The above group of subjects is suffering from TGCT, (b)Schematic diagrams of FAM18B2:p.C51Y,BTN2A3P:p.P3S,MUC2:pG 1715S,NBPF10:p.L44V,SP8:p.G156S,DCP1B:p.Q252H,DEK:p.E41D,ERC1: p.K692R,FAM104B:p.D75H,FRG1B:p.M49V,KRTAP10-10:p.V234M,LRRCC1:p.A6V,NRAS:p.Q61R,PNPLA4:p.L223P,ANkle1:p.C644fs,ANkle1:p.C644 fs,KIT:p.D816H,KIT:p.D816Y,MUC2:p.T1597I,PSMD11:p.A5V,RHPN2:p.V73M,RUNX2:p.Q71E,SP4:p.E7K,TUBA1C:p.L146F,ZNF814:p.Y324H,ADA MTS17:p.N572T,ATRX:p.K1936R,BCL11B:p.E535D,BMP2K:p.Q460H,BMP2K:p.H487Q,C12orf32:p.D60V,C22orf43:p.K19E,CDC27:p.N571I,CDC27:p .P242S,DDX11:p.K208fs,EBPL:p.L189V,EZH2:p.K510R,FAM86A:p.A141T,GAS2L2:p.D189A,GRID2IP:p.LS754del,HGC6.3:p.E171G,KIT:p.D816V ,KIT:p.N822Y,KIT:p.N822K,KRAS:p.Q61R,KRAS:p.G12V,KRTAP1-1:p.I116V,LRRC37BP1:p.Y166D,MEF2A:p.R127Q,MFF:p.S7F,MST1:p.R347W,MUC 4:p.S3048L,MUC6:p.H2000Q,MUC6:p.P1977H,NAT10:p.I393T,OPLAH:p.A900D,PIEZO1:p.Q749E,PRAMEF4:p.F300V,RBM10:p.E184D,SERINC2:p.T1 21P,SPIN2A:p.M150V,SRRM2:p.A2257S,SSBP3:p.K6R,ZNF680:p.R501W,ABCC8:p.Y512C,ABCC9:p.L466P,ABCD1:p.H169Q,ABL2:p.P19T,ACVR2B:p.R48C,AHDC1:p.P33fs,AHNAK2:p.L1640M,ALPPL2:p.W31S,AMMECR1:p.G77C,ANK3:p.D1322E,ANKHD1-EIF4EBP3:p.G60S,ANKRD11:p.Y2015S,ANKRD11:p.K369R,ANKRD50:p.V637M,APBB3:p.L450P,ARHGAP24:p.T35A,ARID4B:p.G1076A,ARMC3:p.A514T,ARRB2:p.T99P,ATAD5:p.I305V,ATXN3:p.305_306insQQQQQQQ,AVPR1B:p.G39R,AXDND1:p.E994Q,BAI2:p.A231G,BEST3:p.P383L,BIRC6:p.V414L,BIRC8:p.A225M,BRWD1:p.K1319R,BTN2A2:p.L15F,C12orf51:p.A2644T,C12orf65:p.K143T,C16orf62:p.L244I,C1QBP:p.T225I,C1orf167:p.S123G,C5orf25:p.Y4F,CACNA1E:p.G2080S,CAPNS1:p.LV303del,CCDC159:p.A332S,CDKAL1:p.P409L,CDYL:p.V48A,CDYL:p.A60G,CELSR2:p.L17P,CHD4:p.E138D,CKAP5:p.G576A,CLCC1:p.K52R,CMTM8:p.S26T,CNKSR2:p.P249L,CNTN5:p.I501T,COG5:p.H617R,COL15A1:p.K708R,COL6A3:p.A2378D,CRYGB:p.R143G,CSGALNACT2:p.L362F,CUL4A:p.I438F,CXXC1:p.Q156H,CYP19A1:p.F406L,DCLRE1B:p.F28I,DDX11:p.A376T,DDX11:p.E680D,DEPDC5:p.R1525Q,DLC1:p.S741T,DNMT1:p.R995Q,DOCK11:p.Q169E,DSPP:p.D. 1047N,E2F7:p.I91S,EBF1:p.D353G,ECI2:p.K55R,EEF1A2:p.Y418S,EIF3J:p.A8G,EML6:p.K805R,EPAS1:p.S474T,EPRS:p.L1335I,ERICH1:p.E327K,FAM101B:p.L5P,FAM104A:p.M1R,FAM110D:p.R71H,FAM155A:p.Q95R,FAM186A:p.G1492E,FAM194B:p.Y139H,FAM21B:p.P1231S,FAM32A:p.K9R,FAM46B:p.H416R,FAM48B1:p.I499V,FAM48B1:p.A516P,FAM5C:p.S425W,FAM86C2P:p.C120Y,FBXL14:p.V48G,FRMPD3:p.Q832del,FRS2:p.L47S,GDF5:p.E105fs,GPNMB:p.C3fs,GPT2:p.R10P,H2AFV:p.Q125R,HDLBP:p.R503C,HERC2:p.R2129C,HIST1H2BJ:p.K13R,HLX:p.N231K,HMGB3:p.E198D,HSF4:p.R169W,HSF4:p.S491P,HYAL4:p.D222N,INO80E:p.P206fs,INTS4:p.S460A,IQCF6:p.R3H,ITPR1:p.M1569I,ITPR3:p.R1698G,KANSL3:p.G376E,KCNA4:p.E627del,KDM5A:p.P423S,KDM6A:p.Y362fs,KIAA0020:p.K63R,KIDINS220:p.N851S,KIT:p.W557G,KLHDC2:p.W321S,KRAS:p.A146T,KRAS:p.Q61H,KRAS:p.Q61L,KRAS:p.G12A,KRAS:p.G12R,KRBA1:p.R839G,KRTAP4-8:p.T63S,L2HGDH:p.P441del,LAMC3:p.P174Q,LHCGR:p.L16Q,LOC401296:p.L144M,LPHN2:p.F906I,LRP12:p.G310C,LTB4R:p.F73L,LTBP3:p.L35del,LUC7L3:p.S148T,LYPD4:p.T64K,MAMLD1:p.Q572L,MAP4K2:p.R341G,MAPK7:p.A501D,MAT2A:p.E166G,MED12L:p.C1292Y,MESP2:p.Q182E,MEX3C:p.R534S,MIER2:p.L131F,MLL5:p.Y66C,MLLT3:p.177_178SS>S,MMS19:p.D1005N, MRPS25:p.E119del,MSH6:p.D576A,MTIF3:p.G65E,MUC17:p.M1807T,MUC17:p.T2279N,MUC17:p.G2474S,MUC2:p.TTPSPP1475del,MUC2:p.T1568M,MU C2:p.T1580N,MUC2:p.T1704I,MUC2:p.T1706M,MUC4:p.H1117D,MUC5B:p.R1097H,MYEF2:p.K323E,MYEOV:p.L302H,MYH8:p.A785V,MYO1A:p.N584K,N AP1L3:p.P353R,NAV1:p.I1433M,NCAM1:p.E131G,NEB:p.D3107N,NEFH:p.V670E,NELL2:p.G170D,NHS:p.D1561N,NKD2:p.H447del,NSD1:p.T461R,NT 5C3:p.A3P,NYAP1:p.P480S,OBSCN:p.A908T,OR10J1:p.R244Q,OR1S2:p.M298I,OR2L3:p.K294R,OR6K6:p.F311L,PABPC3:p.V325fs,PBX2:p.Y262F,PCDHB4:p.P255F,PCMTD1:p.V281A,PCP4L1:p.K64R,PDE3A:p.A98E,PDIA6:p.N56K,PDS5A:p.L1309F,PHLDA2:p.R28S,PIGR:p.V183G,PIK3CA:p.E545K ,PIK3CD:p.C381R,PKD1:p.T938M,PLEKHM1:p.A895V,PLEKHN1:p.A600D,PLXND1:p.R367L,PMS2:p.K651R,PNMA3:p.E200G,POTEF:p.S112G,PRAMEF8: p.I448V,PRDM2:p.E278D,PRODH:p.L527V,PRPF31:p.R289W,PSME4:p.N495D,PTGR1:p.E40A,PTPRB:p.Q726H,RABGEF1:p.N207D,RAC1:p.P34R,RANBP.17:p.M900I,REV3L:p.A30S,RFC3:p.I82N,RFC3:p.K296N,RIMBP3:p.Q1154R,RPL19:p.R151C,RPL5:p.R58fs,RPTN:p.M538I,RRAD:p.A278E,RYR1:p.D668Y,RYR2:p.L2023F,SAFB:p.G799V,SCRIB:p.G332V,SDK1:p.Y2146C,SEC16A:p.T443K,SEC31B:p.P905S,SELO:p.R565Q,SELP:p.A297T,SI:p.I1681K,SLC2A7:p.H268Q,SLC37A1:p.V528I,SLC38A1:p.G100R,SMARCA2:p.D1158A,SMARCA5:p.T156fs,SMC3:p.E970Q,SMG1:p.P2696H,SNRNP200:p.A2129G,SPIN2B:p.M150V,ST6GALNAC1:p.S354N,STAMBPL1:p.Y143H,STARD8:p.G662A,STON1-GTF2A1L:p.N451S,SYMPK:p.A336G,TAS2R8:p.W98C,TCHH:p.W1016R,TET1:p.T1472S,TIAM1:p.G247M,TNS1:p.P183S,TOR1AIP2:p.G146R,TPRX1:p.S216P,TPRX1:p.S200P,TRMT61A:p.S244I,TSPAN4:p.L92V,TTF1:p.Q530R,UBE2M:p.G131D,UBR5:p.R2517S,UGT2B11:p.R447I,UMODL1:p.M559I,UNC93A:p.V445A,USP46:p.Q137R,VWA2:p.G317D,VWA7:p.V792G,WASH3P:p.L187V,WNT5B:p.K327E,WRN:p.E510D,XDH:p.P410S,ZAN:p.S755P,ZC3H11A:p.I777T,ZC3H7A:p.C575S,ZDHHC11:p.H250Q,ZFHX4:p.D3239N,ZKSCAN3:p.K200A,ZMYM4:p.T367I,ZNF174:p.P353T,ZNF322:p.Y353C,ZNF592:p.K324Q,ZNF592:p.P500T,ZNF782:p.C145F,ZNF799:p.This includes any combination of mutations selected from the group consisting of C453R, ZNF804B:p.P644S, and ZNRF3:p.R889W. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0120] 70.(a) The above group of subjects is suffering from THCA, (b) At least one of the above tumor-specific mutations is BRAF:p.V600E,NRAS:p.Q61R,HRAS:p.Q61R,NRAS:p.Q61K,OTUD4:p.T909I,HRAS:p.Q61K,NLRP6:p.E611G,AKT1:p.E17K,ANKMY1:p.N302I,ATP6V1A:p.L237P,CYP19A1:p.S113I,DCUN1D4:p.L275P,DGCR8:p.E518K,DLC1:p.S741T,DNAH10:p.C1853F,EIF1AX:p.G9 This includes any combination of mutations selected from the group consisting of D,FAM75D5:p.L222P,FCGRT:p.P40A,KRAS:p.Q61K,LMX1B:p.Q285del,MAS1L:p.R324G,MED15:p.S35I,MEGF6:p.Y393C,ODZ2:p.A1529V,OR5L1:p.R122H,OR6K6:p.F311L,OTX1:p.D315N,POTEE:p.S75G,SCN5A:p.D1978H,TOP2A:p.K1199E, and TSG101:p.K265R. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0121] 71. (a) The above group of subjects is suffering from UCS, (b) At least one of the above tumor-specific mutations is TP53:p.R248Q, ZNF814:p.D404E, BTN2A3P:p.P3S, FBXW7:p.R465C, FRG1B:p.G65E, MUC4:p.H4205Q, NBPF10:p.V99F, PIK3CA:p.E545K, PIK3CA:p.H1047R, PPP2R1A :p.P179R,DDX11L2:p.*128Q,FBXW7:p.R479Q,FRG1B:p.K13N,FRG1B:p.L52S,HSD17B7P2:p.N175S,KRAS: p.G12V,LOC283788:p.S37G,TP53:p.R273H,TP53:p.S241Y,ADAMTS12:p.E359K,BCL2L11:p.L187fs,CDC2 7:p.L460fs,CHEK2:p.K373E,ESPNP:p.W122fs,FBXW7:p.R689W,FBXW7:p.R505G,FBXW7:p.R465H,FCGBP: p.V4019M,FRG1B:p.I10T,FRG1B:p.D32V,FRG1B:p.R37K,KRAS:p.G12D,LOC100233156:p.R21C,LOC283788 :p.I46M,LRP1B:p.L1392F,MAMLD1:p.Q572L,MST1P9:p.L319P,MUC4:p.A2390T,MUC4:p.G2172S,NBPF10: p.E3455K,PIK3CA:p.G106V,PODXL:p.28_30PSP>P,POTEC:p.R477Q,PPP2R1A:p.R183W,PPP2R1A:p.S219L, This includes any combination of mutations selected from the group consisting of PTPN18:p.TG378del, RGPD3:p.N756D, RPL13AP20:p.G107R, SAMD4B:p.R477W, SMAP1:p.E169fs, TP53:p.H193R, TP53:p.H179R, TP53:p.R175H, TUBBP5:p.R119H, and U2AF1:p.S34F. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0122] 72.(a) The above group of subjects suffers from PAAD, (b)Schematic diagrams of 1 and 1 are RBM14:p.AAAAAAA286del,KRAS:p.G12D,JMY:1. p.PPPPPPPPPPPP811part,RIOK1:p.D69part,LCE2A:p.SSGGCCGSSSGGCC47de l,KRAS:p.G12V,C1QB:p.GPKGPMGPKGGPGAPGAP90part,ZFHX3:p.V777part,DBR1:p.541_542DD>D,AEBP1:p.K1133part,KRAS:p.G12R,RBM47:p.495_502A AAAAAAA>A,AP3S1:p.K41fs,MLL2:p.AEGPHLSPQPEELHLSPQ792part,RFX1:p.386_401GGGGGGGGGGGGGSG>G,AXDND1:p.EQ991part,HERC2P3:p.A803V,R GPD3:p.N756D,FNDC1:p.D1180part,ANAPC1:p.T537A,IRS4:p.21_22AA>A,GIGYF2:p.Q1005part,NCOA3:p.Q1253fs,SIK3:p.950_951QQ>Q,GPR6:p.AAA AATAAGGPDTGEWGPPA36part,NBPF12:p.D1323fs,SHROOM4:p.1156_1157EE>E,ZMIZ2:p.VAAAAATATATAT153part,DGKK:p.PAPP41part,LZTS1:p.RTQDL EGALRTKGL432del,CASQ2:p.395_396DD>D,DCP1B:p.251_252insH,ESPNP:p.296_317PPPPSFPPPPPPPGTQLPPPPP>P,KBTBD6:p.T403K,NBPF16:p.D44 9fs,ANKRD36C:p.H438R,ESPN:p.PPPPPPSFPPPPPPPGTQLPP430part,FCGBP:p.A2493V,KRAS:p.Q61H,NCOA3:p.Q1276part,OR2T2:p.C203fs,TMCC1:p.Q5 65L,BCKDHA:p.G129fs,ESPNP:p.H64fs,GNAS:p.R844H,NBPF14:p.R25C,OGFOD1:p.G477fs,RBM12:p.P693S,SLC38A10:p.1071_1072II>I,SORBS2:p.This includes any combination of mutations selected from the group consisting of P866S, TP53:p.R248W, TP53:p.R175H, and UBAC1:p.E269del. A pharmaceutical composition as described in any one of paragraphs 30 to 36.

[0123] 73. The pharmaceutical composition according to any one of paragraphs 30 to 72, 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 neoantigen peptides.

[0124] 74. The pharmaceutical composition according to paragraph 73, wherein the composition comprises 15 to 20 neoantigen peptides.

[0125] 75. The pharmaceutical composition according to paragraph 73 or 74, further comprising at least one additional neoantigen peptide that is specific to the tumor of an individual patient.

[0126] 76. The pharmaceutical composition according to paragraph 75, wherein a neoantigen peptide specific to the above patient is selected by identifying sequence differences between the genome, exome, and / or transcriptome of a tumor sample from the above patient and the genome, exome, and / or transcriptome of a non-tumor sample.

[0127] 77. The pharmaceutical composition of paragraph 75, wherein the sample is fresh or formalin-fixed paraffin-embedded tumor tissue, newly isolated cells, or circulating tumor cells.

[0128] 78. The pharmaceutical composition according to paragraph 75, wherein the above sequence differences are determined by next-generation sequencing.

[0129] 79. A pharmaceutical composition according to any one of paragraphs 30 to 78, wherein each neoantigen peptide is approximately 5 to approximately 50 amino acids long.

[0130] 80. The pharmaceutical composition according to paragraph 79, wherein each neoantigen peptide is approximately 15 to approximately 35 amino acids long, approximately 15 amino acids or less long, approximately 8 to approximately 11 amino acids long, or 9 or 10 amino acids long.

[0131] 81. The pharmaceutical composition according to paragraph 79 or 80, wherein each neoantigen peptide is bound to major histocompatibility complex (MHC) class I.

[0132] 82. Each neoantigen peptide binds to MHC class I with a binding affinity of less than approximately 500 nM, or, optionally, each neoantigen peptide has a binding affinity of less than 500 nM. D A pharmaceutical composition according to any one of paragraphs 30 to 81, wherein the HLA-A, -B, or -C is bound to the HLA-A, -B, or -C.

[0133] 83. The pharmaceutical composition according to paragraph 79, wherein each neoantigen peptide is approximately 30 amino acids or less in length, approximately 6 to approximately 25 amino acids in length, approximately 15 to approximately 24 amino acids in length, or approximately 9 to approximately 15 amino acids in length.

[0134] 84. The pharmaceutical composition according to paragraph 79, 82, or 83, wherein each neoantigen peptide is bound to major histocompatibility complex (MHC) class II.

[0135] 85. Each neoantigen peptide binds to MHC class I with a binding affinity of less than approximately 500 nM, or, optionally, each neoantigen peptide has a binding affinity of less than 500 nM. D The pharmaceutical composition according to paragraph 84, wherein the HLA-A, -B, or -C is bound to the HLA-A, -B, or -C.

[0136] 86. A pharmaceutical composition according to any one of paragraphs 30 to 85, wherein at least one neoantigen peptide further comprises a flanking amino acid.

[0137] 87. The above flanking amino acid is not a natural flanking amino acid, as described in paragraph 86 of the pharmaceutical composition.

[0138] 88. A pharmaceutical composition according to any one of paragraphs 30 to 87, wherein at least one neoantigen peptide is linked to at least a second neoantigen peptide.

[0139] 89. The pharmaceutical composition according to paragraph 88, wherein the peptides are linked using polyglycine or polyserine linkers.

[0140] 90. The pharmaceutical composition according to paragraph 88 or 89, wherein the second neoantigen peptide binds to MHC class I or class II with a binding affinity of less than 1000 nM.

[0141] 91. The pharmaceutical composition according to any one of paragraphs 88 to 90, wherein the second neoantigen peptide described above binds to MHC class I or class II with an affinity of less than approximately 500 nM.

[0142] 92. A pharmaceutical composition according to any one of paragraphs 88 to 91, wherein both neoepitopes bind to human leukocyte antigen (HLA)-A, -B, -C, -DP, -DQ, or -DR.

[0143] 93. The pharmaceutical composition according to any one of paragraphs 88 to 92, wherein the isolated neoantigen peptide and the second neoantigen peptide bind to class I HLA, or the isolated neoantigen peptide and the second neoantigen peptide bind to class II HLA.

[0144] 94. The pharmaceutical composition according to any one of paragraphs 88 to 92, wherein the isolated neoantigen peptide binds to class II HLA and the second neoantigen peptide binds to class I HLA, or the isolated neoantigen peptide binds to class I HLA and the second neoantigen peptide binds to class II HLA.

[0145] 95. A pharmaceutical composition according to any one of paragraphs 30 to 94, further comprising modifications of at least one neoantigen peptide to increase in vivo half-life, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.

[0146] 96. The pharmaceutical composition according to paragraph 95, wherein the above modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylated HES, recombinant PEG mimetic, Fc fusion, albumin fusion, attachment of nanoparticles, encapsulation of nanoparticles, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, oxidation of side chains, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimetic, or addition of non-natural amino acids.

[0147] 97. The pharmaceutical composition according to paragraph 95, wherein the target cells are antigen-presenting cells.

[0148] 98. The pharmaceutical composition according to paragraph 97, wherein the antigen-presenting cells are dendritic cells.

[0149] 99. The pharmaceutical composition according to paragraph 98, wherein the above-mentioned dendritic cells are targeted using DEC205, XCR1, CD197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD11c, CD83, TSLP receptor, or CD1a marker.

[0150] 100. The pharmaceutical composition according to paragraph 99, wherein the dendritic cells described above are targeted using the CD141, DEC205, or XCR1 marker.

[0151] 101. A pharmaceutical composition according to any one of paragraphs 30 to 100, which is an immunogenic composition or a vaccine composition.

[0152] 102. The pharmaceutical composition according to paragraph 101, further comprising an immunomodulator or adjuvant.

[0153] 103. The above immunomodulatory drugs or adjuvants include poly(I:C), polyICLC, STING agonist, 1018 ISS, 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 A pharmaceutical composition as described in paragraph 102, selected from the group consisting of VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel®, vector system, PLG microparticles, reximod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, acrylic polymer or methacrylic polymer, maleic anhydride copolymer, and QS21 stimulon.

[0154] 104. An isolated polynucleotide encoding an isolated neoantigen peptide as described in any one of paragraphs 1 to 24.

[0155] 105. An isolated polynucleotide, which is RNA, as described in paragraph 104.

[0156] 106. The above RNA has been modified to have increased stability, increased cell targeting, increased translation efficiency, increased adjuvant activity, increased cytosolic reach, and / or decreased cytotoxicity, as described in paragraph 105 of the isolated polynucleotide.

[0157] 107. The isolated polypeptide described in paragraph 106, wherein the modifications include conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC content, incorporation of a modified nucleotide, incorporation of a 5'-cap or cap analogue, and / or incorporation of an exposed polyA sequence.

[0158] 108. Cells containing polynucleotides as described in any one of paragraphs 104-107.

[0159] 109. A vector containing a polynucleotide as described in any one of paragraphs 104-107.

[0160] 110. The vector described in paragraph 110, wherein the above polynucleotides are operably ligated to a promoter.

[0161] 111. A vector as described in paragraph 109 or 110, which is a plasmid, phage, transposon, cosmid, virus, or viral particle.

[0162] 112. Adeno-associated viruses, herpesviruses, lentiviruses or their related viruses The vector described in paragraph 111 is a type.

[0163] 113. An in vivo delivery system containing an isolated polynucleotide as described in any one of paragraphs 104-107.

[0164] 114. The delivery system described in paragraph 113, wherein the delivery system comprises a globular nucleic acid, a virus, a virus-like particle, a plasmid, a bacterial plasmid, or nanoparticles.

[0165] 115. Cells containing a vector or delivery system described in any one of paragraphs 109-114.

[0166] 116. Antigen-presenting cells, as described in paragraph 115.

[0167] 117. Dendritic cells, as described in paragraph 116.

[0168] 118. Immature dendritic cells, as described in paragraph 117.

[0169] 119. A composition comprising at least one polynucleotide as described in any one of paragraphs 104 to 107.

[0170] 120. The composition according to paragraph 119, wherein 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 of the isolated polynucleotides.

[0171] 121. The composition according to paragraph 120, wherein the composition comprises about 2 to about 20 polynucleotides.

[0172] 122. The composition according to any one of paragraphs 119 to 121, further comprising an additional neoantigen polynucleotide encoding an additional neoantigen peptide, 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.

[0173] 123. The composition according to paragraph 122, comprising about 4 to about 20 additional neoantigen polynucleotides.

[0174] 124. The composition according to paragraph 122, wherein the isolated polynucleotide and the additional neoantigen polynucleotide are linked together.

[0175] 125. The composition according to paragraph 124, wherein the polynucleotides are linked using nucleic acids encoding polyglycine or polyserine linkers.

[0176] 126. At least one of the above additional neoantigen peptides is specific to the tumor of the individual patient. A composition that is different from any one of paragraphs 122 to 125.

[0177] 127. The composition according to paragraph 126, wherein a neoantigen peptide specific to the patient is selected by identifying sequence differences between the genome, exome, and / or transcriptome of a tumor sample from the patient and the genome, exome, and / or transcriptome of a non-tumor sample.

[0178] 128. The above sample is fresh or formalin-fixed paraffin-embedded tumor tissue, newly isolated cells, or circulating tumor cells, the composition of paragraph 127.

[0179] 129. The composition according to paragraph 127 or 128, wherein the above sequence differences are determined by next-generation sequencing.

[0180] 130. A T cell receptor (TCR) that can bind to at least one neoantigen peptide listed in any one of paragraphs 1-27, and optionally to a neoantigen peptide including FGFR3 S249C, ERBB3 V104M, EGFR L858R, MUC4 H4205Q, PDGFRA R483fs, TMEM52 23_26LLPL>L, or PODXL 28_30PSP>P.

[0181] 131. The TCR described in paragraph 130, which can bind to the isolated neoantigen peptide in association with MHC class I or class II.

[0182] 132. A chimeric antigen receptor comprising (i) a T cell activating molecule, (ii) a transmembrane domain, and (iii) an antigen recognition portion capable of binding to any one of the isolated neoantigen peptides described in paragraphs 1 to 27.

[0183] 133. CD3-zeta is a T cell activating molecule, a chimeric antigen receptor as described in paragraph 132.

[0184] 134. A chimeric antigen receptor according to paragraph 132 or 133, further comprising at least one co-stimulatory signaling domain.

[0185] 135. A chimeric antigen receptor as described in any one of paragraphs 132-134, wherein the above signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon RI-gamma.

[0186] 136. A chimeric antigen receptor according to any one of paragraphs 132 to 135, wherein the antigen recognition portion can bind to the isolated neoantigen peptide in association with MHC class I or class II.

[0187] 137. A chimeric antigen receptor according to any one of paragraphs 132 to 136, comprising CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane domain.

[0188] 138. The tumor-specific epitope is located in the extracellular domain of a tumor-associated polypeptide, and optionally, the tumor-specific epitope is a chimeric antigen receptor as described in any one of paragraphs 132 to 137, comprising FGFR3 S249C, ERBB3 V104M, EGFR L858R, MUC4 H4205Q, PDGFRA R483fs, TMEM52 23_26LLPL>L, or PODXL 28_30PSP>P.

[0189] 139. T cells containing a T cell receptor or chimeric antigen receptor as described in any one of paragraphs 130-138.

[0190] 140. A T cell as described in paragraph 139, which is a helper or cytotoxic T cell.

[0191] 141. A nucleic acid comprising a promoter operably ligated to a polynucleotide encoding a T cell receptor as described in paragraph 130 or 131.

[0192] 142. The nucleic acid described in paragraph 141, wherein the TCR can bind to at least one neoantigen peptide in association with major histocompatibility complex (MHC) class I or class II.

[0193] 143. A nucleic acid comprising a promoter operably ligated to a polynucleotide encoding a chimeric antigen receptor as described in any one of paragraphs 132-138.

[0194] 144. The nucleic acid described in paragraph 143, wherein the antigen recognition portion can bind to at least one neoantigen peptide in association with a major histocompatibility complex (MHC) class I or class II.

[0195] 145. The nucleic acid described in paragraph 143 or 144, wherein the tumor-specific epitope is located in the extracellular domain of the tumor-associated polypeptide.

[0196] 146. A nucleic acid as described in any one of paragraphs 143 to 145, comprising CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.

[0197] 147. An antibody capable of binding to at least one neoantigen peptide listed in Tables 1-9.

[0198] 148. Modified cells that have been transfected or transduced with any of the nucleic acids described in paragraphs 141-146.

[0199] 149. The modified cells described above are T cells, tumor-infiltrating lymphocytes, NK-T cells, TCR-expressing cells, CD4+ T cells, CD8+ T cells, or NK cells, as described in paragraph 148.

[0200] 150. A composition comprising a T cell receptor or a chimeric antigen receptor as described in any one of paragraphs 130 to 138.

[0201] 151. A composition comprising an autologous patient T cell containing a T cell receptor or chimeric antigen receptor as described in any one of paragraphs 130 to 138.

[0202] 152. The composition according to paragraph 150 or 151, further comprising an immune checkpoint inhibitor.

[0203] 153. The composition according to paragraph 150 or 151, further comprising at least two immune checkpoint inhibitors.

[0204] 154. The above immune checkpoint inhibitors include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, The composition described in paragraph 152 or 153, which inhibits a checkpoint protein selected from the group consisting of VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and B-7 family ligands or combinations thereof.

[0205] 155. The composition described in paragraph 154, wherein the above-mentioned immune checkpoint inhibitor interacts with a ligand for 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.

[0206] 156. A composition according to any one of paragraphs 119-129 or 150-156, further comprising an immunomodulator or adjuvant.

[0207] 157. The composition according to paragraph 156, wherein the immunomodulator is a costimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB.

[0208] 158. The composition according to paragraph 156, wherein the immunomodulator is at least one cancer cell or cancer cell extract.

[0209] 159. The composition according to paragraph 158, wherein the cancer cells are autologous to a subject requiring the composition.

[0210] 160. The composition according to paragraph 159, wherein the cancer cells are lysed or exposed to UV radiation.

[0211] 161. The composition according to paragraph 156, further comprising an adjuvant.

[0212] 162. The above adjuvants are poly(I:C), polyICLC, STING agonist, 1018 ISS, 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 The composition described in paragraph 161, selected from the group consisting of VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel®, vector system, PLG microparticles, reximod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, acrylic polymer or methacrylic polymer, maleic anhydride copolymer, and QS21 stimulon.

[0213] 163. The composition according to paragraph 161 or 162, wherein the adjuvant induces a humoral immune response when administered to a subject.

[0214] 164. The composition described in paragraph 162, wherein the adjuvant induces a T helper cell type 1 response when administered to a subject.

[0215] 165. An in vivo delivery system comprising a pharmaceutical composition described in any one of paragraphs 30 to 103.

[0216] 166. The delivery system described above comprises cell-permeable peptides, nanoparticle encapsulation, virus-like particles, or liposomes, as described in paragraph 165.

[0217] 167. The delivery system according to paragraph 166, wherein the cell-permeable peptide is TAT ​​peptide, herpes simplex virus VP22, transportan, or Antp.

[0218] Cells comprising an isolated neoantigen peptide according to any one of paragraphs 1 to 29.

[0219] 169. The cells according to paragraph 168, which are antigen - presenting cells.

[0220] 170. The cells according to paragraph 169, which are dendritic cells.

[0221] 171. A method of treating, initiating, enhancing or prolonging an anti - tumor response in a subject having cancer, the method comprising administering to the subject a peptide, polynucleotide, vector, composition, antibody or cell according to any one of paragraphs 1 to 164.

[0222] 172. A method of prophylactic cancer treatment, comprising: (a) selecting an anti - cancer agent for a patient in need thereof, wherein the drug is selected from the group consisting of ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF / MEK inhibitors and anti - estrogen therapy; and (b) prophylactically administering to the subject a pharmaceutical composition according to any one of paragraphs 30 to 103, wherein the at least one neoantigen peptide is derived from a drug - resistant mutation associated with the selected anti - cancer agent. A method comprising the above.

[0223] 173. A method of treating or preventing tumors in a population of subjects in need thereof, the method comprising administering to the subjects an agent comprising an extracellular ligand - binding domain that recognizes a tumor - specific neo - epitope comprising a tumor - specific mutation having an incidence of at least 1% in the subjects of the population.

[0224] 174. The method according to any one of paragraphs 171 to 173, wherein the tumor - specific mutation comprises a mutation listed for any population in Table 9.

[0225] 175. The tumor-specific mutation described above is located within a gene containing an extracellular domain, as described in any one of paragraphs 171-173.

[0226] 176. The tumor-specific mutations described above include FGFR3 S249C, ERBB3 V104M, EGFR L858R, MUC4 H4205Q, PDGFRA R483fs, TMEM52 23_26LLPL>L, or PODXL 28_30PSP>P, as described in paragraph 175.

[0227] 177. The method according to paragraph 176, wherein the tumor-specific mutation is located within the extracellular domain.

[0228] 178. The above tumor-specific mutations are FGFR3 S249C or ERBB3 V104 The method described in paragraph 177, including M.

[0229] 179. The method described in any one of paragraphs 171-178, wherein the subject is a human.

[0230] 180. The above subject has cancer, according to the method described in paragraph 179.

[0231] 181. The method according to paragraph 180, wherein the cancers described above are selected from the group consisting of cancers of the genitourinary tract, gynecology, lung, gastrointestinal tract, head and neck, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematological neoplasms, 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-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, squamous cell carcinoma of the head and neck, soft tissue sarcoma, and small cell lung cancer.

[0232] 182. The method according to any one of paragraphs 171 to 181, wherein the subject has undergone surgical resection of the above-mentioned tumor.

[0233] 183. The method according to any one of paragraphs 171 to 182, wherein the peptide, polynucleotide, vector, composition or cell is administered by intravenous, intraperitoneal, intratumoral, intradermal or subcutaneous administration.

[0234] 184. The method according to paragraph 183, wherein the peptide, polynucleotide, vector, composition or cell is administered to an anatomical site that drains into a lymph node basin.

[0235] 185. The method according to paragraph 184, wherein the administration is within a plurality of lymph node basins.

[0236] 186. The method according to any one of paragraphs 183 to 185, wherein the administration is by a subcutaneous or intradermal route.

[0237] 187. The method according to paragraph 183, wherein a peptide is administered.

[0238] 188. The method according to paragraph 187, wherein the administration is intratumoral.

[0239] 189. The method according to paragraph 183, wherein a polynucleotide is administered and optionally RNA is administered.

[0240] 190. The method according to paragraph 189, wherein the polynucleotide is administered intravenously.

[0241] 191. The method according to paragraph 183, wherein the cell is a T cell or a dendritic cell.

[0242] 192. The method according to paragraph 191, wherein the peptide or polynucleotide comprises an antigen-presenting cell targeting moiety.

[0243] 193. The method according to any one of paragraphs 171 to 192, further comprising administering to the subject at least one immune checkpoint inhibitor.

[0244] 194. The method according to paragraph 193, wherein the checkpoint inhibitor is a biological drug or small molecule.

[0245] 195. The method according to paragraph 193 or 194, wherein the checkpoint inhibitor is selected from the group consisting of monoclonal antibodies, humanized antibodies, fully human antibodies and fusion proteins or combinations thereof.

[0246] 196. The method according to any one of paragraphs 193 to 195, 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.

[0247] 197. The method according to any one of paragraphs 193 to 196, wherein the checkpoint inhibitor described above 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.

[0248] 198. The method according to any one of paragraphs 193-197, wherein two or more checkpoint inhibitors are administered.

[0249] 199. The method according to paragraph 198, wherein the checkpoint inhibitor is (i) ipilimumab or tremelimumab and (ii) nivolumab.

[0250] 200. The method according to any one of paragraphs 193 to 199, wherein the above checkpoint inhibitor and the above composition are administered simultaneously or sequentially in any order.

[0251] 201. The method according to paragraph 200, wherein the peptide, polynucleotide, vector, composition, or cells are administered prior to the checkpoint inhibitor.

[0252] 202. The method according to paragraph 200, wherein the peptide, polynucleotide, vector, composition, or cells are administered after the checkpoint inhibitor.

[0253] 203. The method according to paragraph 200, wherein the administration of the above checkpoint inhibitor is continued throughout the entire treatment with neoantigen peptides, polynucleotides, vectors, compositions, or cells.

[0254] 204. The method according to any one of paragraphs 193 to 203, wherein the treatment with the above-mentioned neoantigen peptide, polynucleotide, vector, composition, or cells is administered to a subject that is partially or unresponsive to treatment with a checkpoint inhibitor.

[0255] 205. The method according to any one of paragraphs 193 to 204, wherein the above checkpoint inhibitor is administered intravenously or subcutaneously.

[0256] 206. The method according to paragraph 205, wherein the above checkpoint inhibitor is subcutaneously administered within approximately 2 cm of the administration site of the above composition.

[0257] 207. The method according to paragraph 206, wherein the composition is administered to the same inflow area lymph nodes as the checkpoint inhibitor.

[0258] 208. The method according to any one of paragraphs 171 to 207, further comprising administering an additional therapeutic agent to the subject before, simultaneously with, or after treatment with the peptide, polynucleotide, vector, composition, or cell described above.

[0259] 209. The method according to paragraph 208, wherein the additional agents are chemotherapeutic agents, immunomodulators, immunometabolism modifiers, targeted therapies, radioanti-angiogenic agents, or immunosuppressive agents.

[0260] 210. The method according to paragraph 209, wherein the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an antimetabolitic agent, or an antimitotic agent.

[0261] 211. The method according to paragraph 208, wherein the additional agents are anti-glucocorticoid-induced tumor necrosis factor family receptor (GITR) agonist antibodies or antibody fragments, ibrutinib, docetaxel (docetaxeol), cisplatin, or cyclophosphamide.

[0262] 212. The method described in any one of paragraphs 171-211, which induces a CD4+ T cell immune response.

[0263] 213. The method according to any one of paragraphs 171-212, which induces a CD4+ T cell immune response and a CD8+ T cell immune response.

[0264] 214. A method for stimulating a target immune response, comprising administering an effective amount of a modified cell or composition described in any one of paragraphs 30-103, 108, 115-129, 139, 140, 148-164, and 168-170.

[0265] 215. The method according to paragraph 214, wherein the immune response is a cytotoxic and / or humoral immune response.

[0266] 216. The method described above stimulates the target T cell-mediated immune response, as described in paragraph 214.

[0267] 217. The method described in paragraph 216, wherein the T cell-mediated immune response is targeted at target cells.

[0268] 218. The method according to paragraph 217, wherein the target cells are tumor cells.

[0269] 219. The modified cells described above are transfected or transduced in vivo according to any one of paragraphs 214-218.

[0270] 220. The modified cells described above are transfected or transduced exovivoically, according to any one of the methods described in paragraphs 214-219.

[0271] 221. The modified cells described above are autologous parental T cells, according to any one of the methods described in paragraphs 214-220.

[0272] 222. The method according to paragraph 221, wherein the autologous parental T cells are obtained from a patient who has been administered a neoantigen peptide or nucleic acid vaccine.

[0273] 223. The method according to paragraph 222, wherein the neoantigen peptide or nucleic acid vaccine comprises at least one individualized neoantigen.

[0274] 224. The method according to paragraph 223, wherein the neoantigen peptide or nucleic acid vaccine described above comprises at least one additional neoantigen peptide listed in Tables 1 to 9.

[0275] 225. The patient described above received chemotherapy, immunomodulators, immunometabolism modifiers, targeted therapy, or radiation before and / or during the administration of the neoantigen peptide or nucleic acid vaccine, according to the method described in paragraph 224.

[0276] 226. The method according to any one of paragraphs 222-225, wherein the patient described above receives treatment with at least one checkpoint inhibitor.

[0277] 227. The method according to any one of paragraphs 222-226, wherein the autologous T cells described above are obtained from a patient who has already received at least one course of T-cell therapy containing a neoantigen.

[0278] 228. The method described above further comprises adoptive T-cell therapy, or any one of the methods described in paragraphs 222-227.

[0279] 229. The adoptive T cell therapy described above is the method described in paragraph 228, which includes autologous T cells.

[0280] 230. The autologous T cells described above target tumor antigens, as described in paragraph 229.

[0281] 231. The adoptive T cell therapy described above further comprises allogeneic T cells, as described in paragraph 228 or 229.

[0282] 232. The allogeneic T cells described above are targeted at tumor antigens, as described in paragraph 231.

[0283] 233. The method according to any one of paragraphs 227-231, wherein the adoptive T-cell therapy described above is administered before the checkpoint inhibitor described above.

[0284] 234. A method for evaluating the effectiveness of any one of paragraphs 171 to 213, comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administration of the modified cells; (ii) measuring the number or concentration of target cells in a second sample obtained from the subject after administration of the modified cells; and (iii) determining an increase or decrease in the number or concentration of target cells in the second sample by comparing it with the number or concentration of target cells in the first sample.

[0285] 235. The method according to paragraph 234, wherein the effect of the treatment is determined by monitoring clinical outcomes; increased, enhanced, or prolonged antitumor activity by T cells; an increase in the number of antitumor T cells or activated T cells compared to the number before treatment; B cell activity; CD4 T cell activity; or a combination thereof.

[0286] 236. The method according to paragraph 235, wherein the effect of the treatment is determined by monitoring a biomarker.

[0287] 237. The method according to paragraph 236, 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 type b natriuretic peptide.

[0288] 238. The clinical outcome is the method described in paragraph 235, selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; immune system antitumor response; tumor expansion, recurrence, or spread; or a combination thereof.

[0289] 239. The method according to paragraph 235, wherein the effect of the above treatment is predicted by the presence of T cells, the presence of a gene sign indicating T cell inflammation, or a combination thereof.

[0290] 240. A kit containing a neoantigen therapy drug as described in any one of paragraphs 1 to 164.

[0291] Accordingly, the object of the present invention is that no previously known product, process of manufacturing a product, or method of using a product is included within the scope of the present invention, as the applicants reserve rights and disclose herein any disclaimer of any previously known product, process, or method. Furthermore, it should be noted that the present invention is intended not to include within the scope of the present invention any product, process of manufacturing a product, or method of using a product that does not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (Section 112, first paragraph of the United States Patent Act) or the European Patent Office (EPO) (Section 83 of the EPC), as the applicants reserve rights and disclose herein any previously described product, process of manufacturing a product, or method of using a product. In implementing the present invention, it may be advantageous to comply with Art. 53(c)EPC and Rule 28(b) and (c)EPC. All rights are expressly reserved to expressly disallow any embodiments that are subject to any patent granted to the applicant in the lineage of this application or any other lineage or any prior application of any third party. Nothing herein shall be construed as a promise.

[0292] In this disclosure and especially in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have meanings pursuant to U.S. patent law; for example, they may mean “includes,” “included,” and “including”; and terms such as “consisting essentially of” and “consists essentially of” may have meanings pursuant to U.S. patent law, for example, they may allow elements not expressly described, but should be noted that they exclude elements found in the prior art or elements that affect the fundamental or novel features of the present invention. Nothing here shall be construed as a promise.

[0293] These and other embodiments are disclosed or evident from the following detailed description and are incorporated herein. [Modes for carrying out the invention]

[0294] To aid in understanding the present invention, several terms and phrases are defined herein.

[0295] Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerances in the art, for example, within two standard deviations of the mean. “About” can be understood to mean within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “about.”

[0296] Unless otherwise specifically stated or evident from the context, the terms used herein are The term "or" is understood to be inclusive. Unless otherwise specifically stated or evident from the context, the terms "a," "an," and "the" are understood to be singular or plural when used herein.

[0297] All gene nomenclature symbols refer to genes commonly known in the relevant art. Gene nomenclature symbols may also be those referred to by the HUGO Gene Nomenclature Committee (HGNC). Any reference to this gene nomenclature symbol refers to the entire gene or a variant of this gene. The HUGO Gene Nomenclature Committee is involved in providing human gene nomenclature guidelines and approving names and symbols for novel and unique human genes. All human gene names and symbols can be searched on the HGNC website, www.genenames.org, and the guidelines for creating these names and symbols are available on this website (www.genenames.org / guidelines).

[0298] "Drug" means any small molecule chemical compound, antibody, nucleic acid molecule, polypeptide, or fragment thereof.

[0299] "To improve" means to reduce, suppress, weaken, decrease, stop, or stabilize the onset or progression of a disease (e.g., neoplasm, tumor, etc.).

[0300] "Modification" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by standard methods known in the art, such as those described herein. As used herein, modification includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change.

[0301] An "analog" refers to a molecule that is not identical but possesses similar functional or structural characteristics. For example, a tumor-specific neoantigen polypeptide analog retains the biological activity of the corresponding naturally occurring tumor-specific neoantigen polypeptide while possessing specific biochemical modifications that enhance the analog's function compared to the naturally occurring polypeptide. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life, for example, without altering ligand binding. Analogs may include non-natural amino acids.

[0302] "Combination therapy" is intended to encompass both sequential administration of therapeutic agents (e.g., neoantigen peptides as described herein) (in other words, each therapeutic agent is administered at a different time) and substantially simultaneous administration of these therapeutic agents or at least two of these therapeutic agents. Substantially simultaneous administration can be achieved, for example, by administering to a single capsule having a fixed ratio of each therapeutic agent or to a plurality of single capsules relating to each of these therapeutic agents. For example, one combination of the present invention may include a pooled sample of neoantigen peptides to be administered at the same or different times, or this combination may be formulated as a single simultaneously formulated pharmaceutical composition containing these peptides. As another example, a combination of the present invention (e.g., a pooled sample of tumor-specific neoantigens) may be formulated as separate pharmaceutical compositions that can be administered at the same or different times. As used herein, the term "simultaneously" means the administration of one or more agents at the same time. For example, in certain embodiments, multiple neoantigen peptides are administered simultaneously. Simultaneously includes contemporaneously (in other words, administration during the same period). In certain embodiments, one or more drugs are administered simultaneously at the same time or on the same day. Each therapeutic agent is administered sequentially or substantially simultaneously via oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissue (e.g., nose, mouth, vagina, and rectum), and ocular routes. This can be achieved by any suitable route, including, but not limited to, intravitreal or intraocular administration. Multiple therapeutic agents can be administered via the same or different routes. For example, one component of a particular combination can be administered intravenously, while the other components of this combination can be administered orally. These components can be administered in any therapeutically effective order. The term "combination" encompasses a group of compounds or non-pharmacological therapies useful as part of a combination therapy.

[0303] The term "neoantigen" or "neoantigenic" refers to a class of tumor antigens that arise from tumor-specific mutations that alter the amino acid sequence of proteins encoded by the genome.

[0304] "Neoplasm" means any disease caused by or resulting from an inappropriately high level of cell division, an inappropriately low level of apoptosis, or both. Cancer is an example of a neoplasm. Examples of cancer include, without limitation, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenström macroglobulinemia, heavy chain disease, and solid Tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct (nile) Examples include duct cancer, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Lymphoproliferative disorders are also considered proliferative disorders.

[0305] In the context of this specification, the term “vaccine” is intended to refer to a pool of tumor-specific neoantigen peptides, e.g., at least two, at least three, at least four, at least five, or more neoantigen peptides. “Vaccine” should be understood to mean a composition that produces immunity for the prevention and / or treatment of a disease (e.g., neoplasm / tumor). Accordingly, a vaccine is an antigen-containing agent intended to be used in humans or animals to produce a specific protective and protective substance by vaccination. A “vaccine composition” may include pharmaceutically acceptable excipients, carriers, or diluents.

[0306] The term "pharmaceutically acceptable" means that the use in animals, including humans, has been approved or is expected to be approved by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0307] "Pharmacologically acceptable excipients, carriers, or diluents" refer to excipients, carriers, or diluents that can be administered to a subject together with a drug and, when administered in a dose sufficient to deliver a therapeutic amount of the drug, do not impair the pharmacological activity of the drug and are non-toxic.

[0308] The "pharmaceutically acceptable salts" of the pooled tumor-specific neoantigens as described herein may be acidic or basic salts that are generally considered in the art to be suitable for use in contact with human or animal tissue without excessive toxicity, irritation, allergic reactions, or other problems or complications. Such salts may include inorganic salts of basic residues such as amines. Examples include salts and organic acid salts, as well as alkali salts or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts are not limited to, but include hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanic acid, e.g., acetic acid, HOOC-(CH2) nExamples include salts of acids such as -COOH (wherein n is 0 to 4). Similarly, examples of pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge in the art further pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those published in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). Generally, pharmaceutically acceptable acidic or basic salts can be synthesized from parent compounds containing a basic or acidic moiety by any conventional chemical method. In short, such salts can be prepared by reacting these compounds in the form of free acid or free base with a stoichiometric amount of a suitable base or acid in a suitable solvent.

[0309] "Polypeptide" or "peptide" means a polypeptide that has been isolated from its naturally occurring and associated components. Typically, a polypeptide is isolated when it is absent at least 60% by weight of the naturally bound proteins and naturally occurring organic molecules. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight, of polypeptide. Isolated polypeptides can be obtained, for example, by extraction from a natural source, by expressing recombinant nucleic acids encoding such polypeptides, or by chemically synthesizing proteins. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0310] As used herein, terms such as “prevent,” “preventive,” “prevention,” and “preventive treatment” refer to reducing the likelihood of a disease or condition developing in an individual who is not currently suffering from the disease or condition but is at risk of developing it or is prone to developing it.

[0311] The terms “prime / boost” or “prime / boost dosing regimen” are intended to refer to the sequential administration of a vaccine or immunogenic or immunological composition. A priming dose (priming) is an administration of a first type of vaccine or immunogenic or immunological composition, which may consist of one or more doses. A boost dose is an administration of a second type of vaccine or immunogenic or immunological composition, which may consist of one or more doses, and may include, for example, an annual dose, or may essentially consist of one or more annual doses. In certain embodiments, the administration of a neoplasm vaccine or immunogenic composition constitutes a prime / boost dosing regimen.

[0312] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1 to 50 is any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all numbers, combinations of numbers, or subranges It is understood that this includes decimal values ​​such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from any endpoint of that range are particularly intended. For example, nested subranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0313] The term "receptor" should be understood to mean a biomolecule or molecular classification that possesses ligand-binding ability. Receptors can function to transmit information in cells, cell formation, or organisms. A receptor comprises at least one receptor unit, and often two or more receptor units, where each receptor unit may consist of a protein molecule, more specifically a glycoprotein molecule. A receptor has a structure that complements the structure of the ligand and can form a complex with the ligand as a binding partner. After binding to the ligand on the cell surface, signal information can be transmitted by a change in the three-dimensional structure of the receptor. According to the present invention, a receptor may refer to specific MHC class I and II proteins that have the ability to form a receptor / ligand complex with a ligand, more specifically a peptide or peptide fragment of a suitable length.

[0314] The term "subject" refers to an animal that is the subject of treatment, observation, or experimentation. For example, subjects include, but are not limited to, mammals, such as humans or non-human mammals, such as non-human primates, cattle, horses, dogs, sheep, or cats.

[0315] The terms “treat,” “treated,” “treating,” and “treatment” all refer to the reduction or remission of a disorder and / or related symptoms (e.g., neoplasms or tumors). “Treat” may also mean administering treatment to a subject after the onset or suspected onset of cancer. “Treat” includes the concept of “mitigating,” which refers to reducing the frequency or severity of any cancer-related symptoms or other adverse effects and / or side effects associated with cancer treatment. The term “to treat” also encompasses the concept of “to manage,” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, for example, extending the remission period in a patient who has previously suffered from the disease. It should be understood that treating a disorder or condition does not necessarily require the complete disappearance of the disorder, condition, or related symptoms, although this is not excluded.

[0316] The term “therapeutic effect” refers to the degree of any reduction in one or more symptoms of a disorder (e.g., neoplasm or tumor) or any associated pathology. “Therapeutic effective dose,” as used herein, refers to the amount of a drug administered in a single or multiple doses to a cell or subject that is effective in extending the survival of a patient with such disorder, reducing, preventing, or delaying one or more signs or symptoms of the disorder, beyond what would be expected in the absence of such treatment. “Therapeutic effective dose” is intended to determine the eligibility of the amount required to achieve the therapeutic effect. A physician or veterinarian with ordinary art can readily determine and prescribe the “therapeutic effective dose” (e.g., ED50) of the required pharmaceutical composition. For example, a physician or veterinarian may start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the dose required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0317] The terms "spacer" or "linker," when used in relation to fusion proteins, refer to peptides that bind to proteins containing the fusion protein. Generally, spacers have no specific biological activity other than binding or maintaining a certain minimum distance or other spatial relationship between proteins or RNA sequences. However, in certain embodiments, the constituent amino acids of a spacer may be selected to influence several molecular properties, such as molecular folding, net charge, or hydrophobicity.

[0318] Suitable linkers for use in embodiments of the present invention are known to those skilled in the art, and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In preferred embodiments, the linker is used to separate two neoantigen peptides by a distance sufficient to ensure that each neoantigen peptide folds correctly. Preferred peptide linker sequences employ flexible extended conformations and do not tend to generate regular secondary structures. Typical amino acids in the flexible protein region include Gly, Asn, and Ser. Substantially, any permutation of amino acid sequences containing Gly, Asn, and Ser would be expected to satisfy the above criteria for linker sequences. Other nearly neutral amino acids (e.g., Thr and Ala) can also be used in linker sequences. Further amino acid sequences that can be used as linkers are disclosed in Maratea et al. (1985), Gene 40:39-46; Murphy et al. (1986), Proc. Nat'l. Acad. Sci. USA 83:8258-62; U.S. Patent No. 4,935,233; and U.S. Patent No. 4,751,180.

[0319] Any list of chemical groups in any definition of a variable group as described herein includes the definition of the variable group as any single group or combination of the enumerated groups. Any description of embodiments relating to a variable group or aspect as described herein includes embodiments as any single embodiment or embodiments in combination with any other embodiment or part thereof.

[0320] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0321] The therapies disclosed herein constitute novel methods for treating various types of cancer. The therapies described herein also provide treatment methods that achieve clinical benefits without unacceptable levels of side effects.

[0322] In one embodiment, the present invention relates to a method for treating neoplasms (more specifically tumors) by administering a vaccine or immunogenic composition comprising a plurality of tumor-specific neoantigen peptides to a subject. As described in more detail herein, in some embodiments, the composition provides a specific optimized subset of tumor-specific neoantigens suitable for treating tumors in a high proportion of subjects suffering from cancer. In some embodiments, these tumor-specific neoantigens can bind together to a high overall proportion of HLA allotypes present in the population of the subject.

[0323] The immune system can be classified into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the front line of defense against infection, and the most potentially capable pathogens are rapidly neutralized by this system before they can cause a recognizable infection, for example. The adaptive immune system responds to the molecular structure of invading organisms, called antigens. There are two types of adaptive immune responses: humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted into the body fluid by B cells bind to pathogen-derived antigens, leading to the elimination of pathogens through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells, which have the ability to destroy other cells, are activated. For example, if disease-related proteins are present in cells, these proteins are fragmented into peptides by proteolysis within the cell. Then, specific cellular proteins attach to these thus formed antigens or peptides and transport them to the cell surface, where these antigens or peptides are presented to the body's molecular defense mechanisms, specifically T cells. Cytotoxic T cells recognize these antigens and kill the cells that possess them.

[0324] Molecules that transport and present peptides to the cell surface are called major histocompatibility complex (MHC) proteins. MHC proteins are classified into two types: MHC class I and MHC class II. The structures of these two MHC class proteins are very similar; however, they have very different functions. MHC class I proteins are present on the surface of almost all cells in the body, including many tumor cells. MHC class I proteins are typically loaded with antigens derived from endogenous proteins or pathogens present within the cell, which are then presented to naive or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. MHC class II proteins primarily present peptides from external antigen sources, i.e., processed outside the cell, to T helper (Th) cells. Most peptides to which MHC class I proteins bind originate from cytoplasmic proteins produced in the organism's own healthy host cells and do not usually stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize such self-peptide-presenting MHC molecules of class I are either eliminated in the thymus (central tolerance) or eliminated or inactivated after release from the thymus, i.e., tolerated (peripheral tolerance). MHC molecules have the ability to stimulate an immune response when they present peptides to non-tolerant T lymphocytes. Cytotoxic T lymphocytes have both a T cell receptor (TCR) and a CD8 molecule on their surface. The T cell receptor has the ability to recognize and bind peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that has the ability to bind to specific MHC / peptide complexes.

[0325] Peptide antigens attach to MHC class I molecules within the endoplasmic reticulum via competitive affinity binding before being presented on the cell surface. Here, the affinity of individual peptide antigens is directly related to their amino acid sequence and the presence of specific binding motifs at defined positions within that sequence. If the sequence of such peptides is known, it is possible to manipulate the immune system against infected cells, for example, using peptide vaccines.

[0326] One of the major obstacles hindering the development of curative and tumor-specific immunotherapies is the identification and selection of highly specific and restrictive tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic changes within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), constitute the most tumor-specific class of antigens. Neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to the technical difficulties in their identification, selection of optimized neoantigens, and production of neoantigens for use in vaccines or immunogenic compositions. These problems can be addressed by: Identifying neoplasm / tumor mutations that are present at the DNA level in tumors but not in corresponding germline samples from a high proportion of subjects with cancer; • Analyze identified mutations using one or more peptide-MHC binding prediction algorithms to generate multiple neoantigen T cell epitopes expressed within neoplasms / tumors and binding to a high proportion of the patient's HLA alleles; and To synthesize multiple neoantigen peptides selected from a set of all neoantigen peptides and predictive binding peptides for use in cancer vaccines or immunogenic compositions suitable for treating a high proportion of subjects with cancer.

[0327] For example, converting sequencing information into therapeutic vaccines includes the following: (1) Prediction of mutant peptides that can bind to HLA molecules in a high percentage of individuals. To efficiently select which specific mutations should be used as immunogens, it is necessary to be able to predict which mutant peptides can efficiently bind to the HLA alleles of a high percentage of patients. In recent years, the accuracy of prediction algorithms for major HLA-A and -B alleles has improved with neural network-based learning methods using validated binding and unbinding peptides. (2) Formulating the drug as a long-peptide multi-epitope vaccine. By targeting as many mutant epitopes as possible, the immense capabilities of the immune system are leveraged, opportunities for immune escape through downregulation of specific immunotargeting gene products are prevented, and known inaccuracies of epitope prediction methods are compensated for. Synthetic peptides provide a particularly useful means for efficiently preparing multiple immunogens and rapidly translating mutant epitope identification into effective vaccines. Peptides can be readily chemically synthesized using reagents free from contaminating bacteria or animal matter and can be easily purified. Their small size allows for clear focus on mutant regions of proteins and reduces irrelevant antigen competition from other components (non-mutant proteins or viral vector antigens). (3) Concomitant use with a potent vaccine adjuvant. Effective vaccines require a potent adjuvant to elicit an immune response. As described below, poly-ICLC, which is a TLR3 agonist and the RNA helicase domains of MDA5 and RIG3, exhibits several desirable properties as vaccine adjuvants. These properties include induction of local and systemic activation of immune cells in vivo, production of stimulating chemokines and cytokines, and stimulation of antigen presentation by DCs. Furthermore, poly-ICLC can induce a sustained CD4+ and CD8+ response in humans. Importantly, significant similarities in the upregulation of transcriptional and signaling pathways were observed between subjects vaccinated with poly-ICLC and volunteers who had previously received a highly effective replication-competent yellow fever vaccine. In addition, in a recent Phase 1 study, more than 90% of ovarian cancer patients immunized with poly-ICLC in combination with the NYES0-1 peptide vaccine (in addition to Montanide) showed induction of CD4+ and CD8+ T cells and an antibody response to the peptide. Simultaneously, polyICLC has been extensively tested in more than 25 clinical trials to date, exhibiting a relatively safe toxicity profile. The advantages of the present invention described above are further described herein.

[0328] As described herein, in both animals and humans, mutagenic epitopes are effective in inducing immune responses, and cases of spontaneous tumor regression or long-term survival correlate with CD8+ T cell responses to mutagenic epitopes (Buckwalter and Srivastava PK. "It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer." Seminars in immunology 20:296-300 (2008); Karanikas et al. "High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival." Cancer Res.61:3718-3724(2001); Lennerz et al, "The response of autologous T cells to a human melanoma is dominated by mutated neo-antigens." Proc Natl Acad Sci US A.102:16013(2005)) and the ability to track "immunoediting" in altering the expression of dominant mutated antigens in mice and humans (Matsushita et al, "Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting"). "Immunoediting" Nature 482:400 (2012); DuPage et al, "The expression of tumor-specific antigens underlies cancer immunoediting (Expre There is ample evidence for the "ssion of tumor-specific antigens underlies cancer immunoediting" (Nature 482:405 (2012)); and for Sampson et al., "Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma" (J Clin Oncol. 28:4722-4729 (2010)). In one embodiment, the mutant epitope of a cancer patient is determined.

[0329] Sequencing techniques have revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of its genes. These mutations produce modified proteins ranging from single-amino acid changes (caused by missense mutations) to the addition of long regions of novel amino acid sequences resulting from frameshifts, stop codon read-throughs, or translation of intron regions (novel open-reading frame mutations; neo-ORFs). Unlike native proteins, these mutant proteins are not subject to self-tolerant immunosuppressive effects, making them useful targets for the host's immune response to tumors. Therefore, mutant proteins are more likely to be immunogenic and exhibit higher specificity to tumor cells compared to the patient's normal cells.

[0330] In one embodiment, multiple neoantigen peptides in the composition share affinity for multiple MHC molecules (e.g., MHC molecules that together cover a large portion of the target population). Efficiently selecting which specific mutations should be used as immunogens requires the ability to predict which mutant peptides can efficiently bind to HLA alleles present in the patient population. In recent years, neural network-based learning methods using validated bound and unbound peptides have improved the accuracy of prediction algorithms for major HLA-A and -B alleles. By utilizing recently improved algorithms that predict which missense mutations will produce strongly binding peptides to cognitive MHC molecules, it is possible to identify and prioritize a set of peptides that represent the optimal mutant epitopes (both neoORFs and missenses) for a patient population (Zhang et al, "Machine learning competition in immunology - Prediction of HLA class I binding peptides" J Immunol Methods 374:1(2011); Lundegaard et al, "Prediction of epitopes using neural network based methods" J Immunol Methods 374:26(2011)).

[0331] By targeting as many mutant epitopes as possible in reality, the vast capabilities of the immune system are utilized, opportunities for immune escape through downregulation of specific immunotargeting gene products are prevented, and known inaccuracies of epitope prediction methods are compensated for. Synthetic peptides provide a particularly useful means for efficiently preparing multiple immunogens and rapidly translating the identification of mutant epitopes into effective vaccines or immunogenic compositions. Peptides can be readily chemically synthesized using reagents free from contaminating bacteria or animal substances and can be easily purified. Their small size allows for clear focus on mutant regions of proteins and reduces irrelevant antigen competition from other components (non-mutant proteins or viral vector antigens).

[0332] In one embodiment, the drug formulation is a long peptide multiepitope vaccine or immunogenic composition. Such “long” peptides have been shown to undergo efficient internalization, processing, and cross-presentation in professional antigen-presenting cells such as dendritic cells, and to induce CTLs in humans (Melief and van der Van Van der Van der Van der Van der Van Van der Van der Van Van der Van der Van Van der Van Van der Van Van der Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van Van der Van der Burg, "Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines," Nature Rev Cancer 8:351 (2008). In one embodiment, at least two peptides are prepared for immunization. In a preferred embodiment, 20 or more peptides are prepared for immunization. In some embodiments, the neoantigen peptides are in the range of about 5 to about 50 amino acids in length. In another embodiment, peptides are synthesized that are about 15 to about 35 amino acids in length. In a preferred embodiment, the neoantigen peptides are in the range of about 20 to about 35 amino acids in length.

[0333] Production of tumor-specific neoantigens The present invention is, at least in part, based on the ability to present a pool of tumor-specific neoantigens to a patient's immune system. Those skilled in the art will understand from this disclosure and the knowledge in the art that there are various methods for producing such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be produced either in vitro or in vivo. Tumor-specific neoantigens may be produced in vitro as peptides or polypeptides, which may then be formulated into neoplasm vaccines or immunogenic compositions and administered to a subject. As described in more detail herein, such in vitro production may be carried out by various methods known to those skilled in the art, such as peptide synthesis or expression of peptides / polypeptides from DNA or RNA molecules in any of various bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptides / polypeptides. Alternatively, tumor-specific neoantigens may be produced in vivo by introducing a molecule encoding a tumor-specific neoantigen (e.g., DNA, RNA, viral expression system, etc.) into a subject, and then expressing the encoded tumor-specific neoantigen after introduction. Methods for the in vitro and in vivo preparation of neoantigens are also described herein when they relate to pharmaceutical compositions and methods for the delivery of therapeutic agents.

[0334] In certain embodiments, the present invention includes modified neoantigen peptides. When used herein with respect to neoantigen peptides, the terms “modified,” “modified,” etc., refer to one or more changes that enhance a desired property of the neoantigen peptide, the changes not altering the primary amino acid sequence of the neoantigen peptide. “Modified” includes covalent chemical modifications that do not alter the primary amino acid sequence of the neoantigen peptide itself. Such desired properties include, for example, an extension of the half-life in vivo, increased stability, decreased clearance, alteration of immunogenicity or allergenicity, and the possibility of increased antibody, cell targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. Possible modifications to the neoantigen peptide include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylated HES, recombinant PEG mimetic, Fc fusion, albumin fusion, attachment of nanoparticles, encapsulation of nanoparticles, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, oxidation of side chains, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimetic, or addition of non-natural amino acids.

[0335] The clinical efficacy of protein-based therapeutics is often limited by their short plasma half-lives and susceptibility to protease degradation. Studies of various therapeutic proteins (e.g., filgrastim) have shown that such difficulties can be overcome by modifying polypeptide sequences with various non-protein polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyglycerides). It has been found that this can be overcome by various modifications, such as conjugating or linking to one of the sialcylenes (see, for example, via a linking moiety that is typically covalent to both protein and non-protein polymers (e.g., PEG)). Such PEG-conjugated biomolecules have been found to possess clinically beneficial properties (e.g., better physical and thermal stability, protection from enzymatic degradation, increased solubility, longer in vivo circulating half-life and reduced clearance, decreased immunogenicity and antigenicity, and reduced toxicity).

[0336] PEGs suitable for conjugation into polypeptide sequences are generally water-soluble at room temperature and have the general formula R(O-CH2-CH2) n The general formula has OR, where R is a protecting group such as a hydrogen atom, alkyl group, or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, R generally has 1 to 8 carbon atoms. PEG conjugated to a polypeptide sequence can be linear or branched. Branched PEG derivatives, "star-PEG," and multi-armed PEG are intended by this disclosure. The molecular weight of the PEG used in this disclosure is not limited to any particular range, although certain embodiments have molecular weights from 500 to 20,000 and other embodiments have molecular weights from 4,000 to 10,000.

[0337] This disclosure also intends to describe compositions of conjugates in which multiple PEGs have different n values, and therefore various different PEGs are present in specific ratios. For example, some compositions contain mixtures of n=1, 2, 3, and 4 conjugates. In some compositions, the proportion of n=1 conjugates is 18-25%, the proportion of n=2 conjugates is 50-66%, the proportion of n=3 conjugates is 12-16%, and the proportion of n=4 conjugates is up to 5%. Such compositions can be prepared by reaction conditions and purification methods known in the art. For example, conjugates can be separated using cation exchange chromatography, and then fractions containing conjugates with a desired number of attached PEGs can be identified and purified to exclude unmodified protein sequences and conjugates with other numbers of attached PEGs.

[0338] PEG can be attached to the polypeptides of this disclosure via terminal reactive groups (spacers). These spacers are, for example, terminal reactive groups that mediate the binding between free amino or carboxyl groups of one or more polypeptide sequences and polyethylene glycol. An example of a PEG having a spacer that can be attached to a free amino group is N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide. Another activated polyethylene glycol that can be attached to a free amino group is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuryl chloride. An example of an activated polyethylene glycol that can be attached to a free carboxyl group is polyoxyethylenediamine.

[0339] Conjugation of one or more polypeptide sequences of the present disclosure onto PEGs having spacers can be carried out by various conventional methods. For example, this conjugation reaction can be carried out in solution at a pH of 5 to 10, a temperature of 4°C to room temperature, and for 30 minutes to 20 hours, using a reagent-to-protein molar ratio of 4:1 to 30:1. The reaction conditions can be selected to lead the reaction to primarily produce a desired degree of substitution. Generally, lower temperatures, lower pH (pH=5), and shorter reaction times tend to result in a decrease in the number of attached PEGs, while higher temperatures, neutral to high pH (e.g., pH>7), and longer reaction times tend to increase the number of attached PEGs. This reaction can be stopped using various means known in the art. In some embodiments, the reaction mixture is used. This reaction can be stopped by acidifying the solution and freezing it, for example, at -20°C.

[0340] This disclosure also intends to explore the use of PEG mimics. Recombinant PEG mimics have been developed that confer several more advantageous properties while retaining the characteristics of PEG (e.g., extended serum half-life). As an example, simple polypeptide chains (e.g., including Ala, Glu, Gly, Pro, Ser, and Thr) that can form an extended higher-order structure similar to PEG, already fused to the peptide or protein drug of interest, can be produced by recombinant (e.g., Amunix'XTEN technology; Mountain View, CA). This eliminates the need for additional conjugation steps in the manufacturing process. Furthermore, established molecular biotechnologies allow for control of the side-chain composition of the polypeptide chain, enabling optimization of immunogenicity and manufacturing properties.

[0341] For the purposes of this disclosure, “glycosylation” means broadly the enzymatic process of attaching glycans to proteins, lipids, or other organic molecules. The use of the term “glycosylation” in connection with this disclosure is generally intended to mean the addition or deletion of one or more carbohydrate moieties (by removing an underlying glycosylation site or by chemical and / or enzymatic means) and / or the addition of one or more glycosylation sites that may or may not be present in the natural sequence. In addition, the term includes qualitative changes in the glycosylation of natural proteins, involving changes in the properties and proportions of the various carbohydrate moieties present. Glycosylation can dramatically affect the physical properties of proteins and may also be important for protein stability, secretion, and intracellular localization. Proper glycosylation may be essential for biological activity. In fact, some eukaryotic genes, when expressed in bacteria lacking the cellular processes for glycosylation proteins (e.g., Escherichia coli), result in proteins that are recovered with little or no activity due to the lack of glycosylation.

[0342] The addition of glycosylation sites can be achieved by modifying the amino acid sequence. For example, polypeptides can be modified by adding or substituting one or more serine or threonine residues (in the case of O-linked glycosylation sites) or asparagine residues (in the case of N-linked glycosylation sites). The structures of N-linked and O-linked oligosaccharides and sugar residues found in each type can differ. One type of sugar commonly found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is typically a terminal residue in both N-linked and O-linked oligosaccharides, and its negative charge can confer acidic properties to glycoproteins. Specific embodiments of this disclosure involve generating and using N-glycosylation variants.

[0343] The polypeptide sequences of this disclosure may be optionally modified by changes at the DNA level, specifically by mutating the DNA encoding the polypeptide with pre-selected bases so that codons that translate to desired amino acids are produced. Another means of increasing the number of carbohydrate moieties on the polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.

[0344] Carbohydrate removal can be achieved chemically, enzymatically, or by substituting codons encoding the amino acid residues to be glycosylated. Chemical glycosylation techniques are known, and enzymatic cleavage of the carbohydrate portion on polypeptides can be achieved using various endoglycosidases and exoglycosidases.

[0345] Dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells are commonly used host cells for recombinant glycoprotein production. These cells do not express the enzyme beta-galactoside alpha-2,6-sialyltransferase. Therefore, the sialic acid in the alpha-2,6-linking is not attached to the N-linked oligosaccharide of the glycoprotein produced in this cell.

[0346] This disclosure also envisions the use of polyacylation, which is the conjugation of peptides and proteins to naturally occurring biodegradable α-(2→8)-linked polysialic acid ("PSA"), to improve the stability and in vivo pharmacokinetics of peptides and proteins. PSA is a highly hydrophilic, biodegradable, non-toxic natural polymer that confers a large apparent molecular weight that extends the serum half-life in the blood. In addition, polysialation of various peptide and protein therapeutics significantly reduces proteolysis, preserves in vivo activity, and reduces immunogenicity and antigenicity (see, e.g., G. Gregoriadis et al., Int. J. Pharmaceuticals 300(1-2):125-30). Similar to modifications using other conjugates (e.g., PEG), various techniques for site-specific polysialation are available (see, for example, T. Lindhout et al., PNAS 108(18)7397-7402 (2011)).

[0347] Further suitable components and molecules for conjugation include, for example, thyroglobulin; albumin, e.g., human serum albumin (HAS); tetanus toxoid; diphtheria toxoid; polyamino acids, e.g., poly(D-lysine:D-glutamic acid); rotavirus VP6 polypeptide; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the above.

[0348] The fusion of albumin to one or more polypeptides of this disclosure can be achieved, for example, by genetic engineering such that DNA or a fragment thereof encoding HSA is conjugated to DNA encoding one or more polypeptide sequences. A suitable host can then be transformed or transfected with the fusion nucleotide sequence, for example, in the form of a suitable plasmid, to express the fusion polypeptide. This expression may be achieved in vitro, for example, from prokaryotic or eukaryotic cells, or in vivo, for example, from a genetically modified organism. In some embodiments of this disclosure, the expression of the fusion protein is carried out in a mammalian cell line (e.g., a CHO cell line). Transformation is used herein to refer to the genetic modification of a cell resulting from the direct uptake, integration, and expression of exogenous genetic material (exogenous DNA) taken in from around the cell and through the cell membrane. Transformation occurs spontaneously in some bacterial species, but can also be achieved by artificial means in other cells.

[0349] Furthermore, albumin itself can be modified to extend its cyclic half-life. Fusion of modified albumin to one or more polypeptides can be achieved by the genetic engineering techniques described above or by chemical conjugation, and the resulting fusion molecule will have a half-life exceeding that of the fusion with unmodified albumin (see International Publication No. 2011 / 051489).

[0350] As an alternative to direct fusion, several albumin-binding strategies have been developed, such as albumin binding (acylation) via conjugated fatty acid chains. Since serum albumin is a fatty acid transport protein, these natural ligands with albumin-binding activity are used to extend the half-life of small protein therapeutics. For example, insulin detemir (LEVEMIR), an approved product for diabetes, contains a myristyl chain conjugated to genetically modified insulin, resulting in an insulin analog that acts over a long period.

[0351] Another type of modification involves conjugating (e.g., linking) one or more additional components or molecules, or carrier molecules, at the N-terminus and / or C-terminus of a polypeptide sequence, such as another protein (e.g., a protein with an amino acid sequence heterogeneous to the target protein). Thus, exemplary polypeptide sequences can be generated as conjugates with other components or molecules.

[0352] Conjugate modification may result in polypeptide sequences that retain activity with further or complementary functions or activities of a second molecule. For example, a polypeptide can be conjugated to a molecule to enhance, for instance, solubility, storage, in vivo or storage half-life or stability, reduced immunogenicity, delayed or controlled in vivo release, etc. Other functions or activities include conjugates that reduce toxicity to unconjugated polypeptide sequences, conjugates that target a single cell or organ more efficiently than unconjugated polypeptide sequences, or drugs that further counteract the causes or effects associated with the disorders or diseases described herein (e.g., diabetes).

[0353] Polypeptides can also be conjugated to large, slowly metabolized macromolecules such as proteins; polysaccharides such as Sepharose, agarose, cellulose, and cellulose beads; polymeric amino acids such as polyglutamic acid and polylysine; amino acid copolymers; inactivated viral particles; inactivated bacterial toxins such as diphtheria, tetanus, cholera, and toxins from leukotoxin molecules; inactivated bacteria; and dendritic cells.

[0354] Further candidate components and molecules for conjugation include those suitable for isolation or purification. Specific, non-limiting examples include binding molecules, such as biotin (biotin-avidin specific binding pairs), antibodies, receptors, ligands, lectins, or molecules containing solid supports (e.g., plastic or polystyrene beads, plates or beads, magnetic beads, test pieces, and membranes).

[0355] Conjugates can be separated by charge differences, efficiently separating them into their various molecular weights using purification methods such as cation exchange chromatography. For example, a cation exchange column can be packed, then washed with -20 mM sodium acetate at pH -4, and then eluted with a linear (0 M to 0.5 M) NaCl gradient buffered at approximately 3 to 5.5 pH (e.g., pH -4.5). The contents of the fraction obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectrometry, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.

[0356] In certain embodiments, the amino-terminus or carboxyl-terminus of the polypeptide sequence of this disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to extend the systemic half-life of biologics, and therefore, biologic products may not require frequent administration.

[0357] Fc binds to neonatal Fc receptors (FcRn) in endothelial cells lining blood vessels. Upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, allowing it to remain in circulation for a longer period. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. More recent Fc fusion technologies ligate a single copy of a biopharmaceutical to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc fusion conjugates.

[0358] This disclosure is intended to improve one or more specific aspects that are currently known or will be developed in the future. The use of other modifications of the lipeptides is intended. One such method for extending the cyclic half-life, increasing stability, decreasing clearance, or modifying the immunogenicity or allergenicity of the polypeptides of this disclosure includes modification of the polypeptide sequence by hesylation, utilizing a hydroxyethyl starch derivative linked to another molecule to alter the properties of the molecule. Various forms of hesylation are described, for example, in U.S. Patent Application Publication No. 2007 / 0134197 and U.S. Patent Application Publication No. 2006 / 0258607.

[0359] In vitro peptide / polypeptide synthesis Proteins or peptides can be prepared by any technique known to those skilled in the art, including the expression of proteins, polypeptides, or peptides by standard molecular biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have already been disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, available on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to those skilled in the art. Alternatively, various commercially available protein, polypeptide, and peptide preparations are known to those skilled in the art.

[0360] Peptides can be easily synthesized chemically using reagents that do not contain contaminating bacteria or animal substances (Merrifield RB: "Solid phase peptide synthesis. I. The synthesis of tetrapeptides"). (Synthesis of a tetrapeptide). J.Am.Chem.Soc.85:2149-54,1963). In certain embodiments, neoantigen peptides are prepared by (1) parallel solid-phase synthesis in a multi-channel instrument using homogeneous synthesis and cleavage conditions; (2) purification by column stripping on an RP-HPLC column and rewashing between peptides, but without exchange; followed by (3) analysis by a limited set of assays with the highest informational value. A Good Manufacturing Practice (GMP) footprint can be defined for a set of peptides for an individual patient, and thus a suite switching procedure is only required between peptide synthesis for different patients.

[0361] Alternatively, the neoantigen peptide may be prepared in vitro using a nucleic acid (e.g., polynucleotide) encoding the neoantigen peptide of the present invention. The polynucleotide may be, for example, DNA, cDNA, PNA, CNA, RNA, single-stranded and / or double-stranded, or a natural or stabilized form of polynucleotide, such as a polynucleotide having a phosphorothioate backbone, or a combination thereof, and may or may not contain introns, insofar as it encodes a peptide. In one embodiment, the peptide is prepared by in vitro translation. Many exemplary systems are available to those skilled in the art (e.g., Retic Lysate IVT Kit, Life Technologies, Waltham, MA).

[0362] Expression vectors capable of expressing polypeptides can also be prepared. Expression vectors for various cell types are well known in the art and can be selected without excessive experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, with the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be ligated to appropriate transcriptional and translational regulatory nucleotide sequences recognized by the desired host (e.g., bacteria), however such regulation is generally available in the expression vector. The vector is then introduced into a host bacterium for cloning using standard techniques (e.g., Sa See mbrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0363] Expression vectors containing isolated polynucleotides, as well as host cells containing the expression vectors, are also intended. Neoantigen peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigen peptide. One or more neoantigen peptides of the present invention may be provided by a single expression vector.

[0364] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides containing only the coding sequence of a polypeptide, as well as polynucleotides containing further coding and / or non-coding sequences. Polynucleotides may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; it may be double-stranded or single-stranded, and in the case of single-stranded, it may be a coding strand or a non-coding strand (antisense strand).

[0365] In embodiments, the polynucleotide may include a coding sequence of a tumor-specific neoantigen peptide fused in the same reading frame as a polynucleotide that assists in the expression and / or secretion of polypeptides from host cells (e.g., a leader sequence that functions as a secretory sequence to control the transport of polypeptides from cells). The polypeptide having the leader sequence is a preprotein and may have a leader sequence that is cleaved by host cells to form a mature polypeptide.

[0366] In embodiments, the polynucleotide may include a coding sequence for a tumor-specific neoantigen peptide fused in the same reading frame to a marker sequence, for example, enabling the purification of the encoded polypeptide so that it can then be incorporated into a personalized neobiotic vaccine or immunogenic composition. For example, the marker sequence may be a hexahistidine tag supplied by a pQE-9 vector that provides purification of a mature polypeptide fused with the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Further tags, though not limited to them, include calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tags, Xpress tags, Isopep tags, SpyTag, biotin carboxyl carrier protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose-binding protein tags, Nus tags, Strep tags, thioredoxin tags, TC tags, and Ty tags.

[0367] In one embodiment, the polynucleotide may comprise one or more coding sequences of tumor-specific neoantigen peptides fused in the same reading frame to create a single concatemerized neoantigen peptide construct having the ability to produce multiple neoantigen peptides.

[0368] In certain embodiments, isolated nucleic acid molecules can be provided that have a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% identical to the polynucleotide encoding the tumor-specific neoantigen peptide of the present invention.

[0369] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence is defined as the nucleotide sequence of the polynucleotide being, with respect to the reference sequence, poly The nucleotide sequence is intended to be identical to that of the reference nucleotide sequence, except that it may contain up to 5 point mutations per 100 nucleotides. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may exist individually scattered among the nucleotides in the reference sequence, or as one or more adjacent clusters within the reference sequence, at the amino-terminus or carboxyl-terminus of the reference nucleotide sequence, or somewhere between those terminal positions.

[0370] In practice, whether any particular nucleic acid molecule is at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, version 8 Unix version, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best homology segment between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and a homology gap of up to 5% of the total number of nucleotides in the reference sequence is permitted.

[0371] The isolated tumor-specific neoantigen peptides described herein can be produced in vitro (e.g., in the laboratory) by any preferred method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in a suitable transforming host. In some embodiments, the DNA sequences are constructed by isolating or synthesizing DNA sequences encoding the wild-type protein of interest using recombination techniques. In some cases, site-directed mutagenesis may be used to induce mutations in the sequences and provide functional analogs. For example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA. See 81:5662-5066 (1984) and U.S. Patent Nos. 4,588,585.

[0372] In embodiments, the DNA sequence encoding the target polypeptide can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting codons preferred by the host cell producing the recombinant polypeptide of the target. Standard methods can be applied to the synthesis of the isolated polynucleotide sequence encoding the target isolated polypeptide. For example, a back-translated gene can be constructed using the complete amino acid sequence. Furthermore, DNA oligomers containing nucleotide sequences encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically include a 5' or 3' overhang for complementary assembly.

[0373] After assembly (e.g., by synthesis, site-directed mutagenesis, or other method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into the expression vector. The gene is then operably ligated to an expression regulatory sequence suitable for the expression of the protein in the desired host. The suitability of the assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to achieve high expression levels of the transfected gene in the host, the gene can be operably ligated to transcriptional and translational expression regulatory sequences that function in a selected expression host.

[0374] Recombinant expression vectors may be used to amplify and express DNA encoding tumor-specific neoantigen peptides. Recombinant expression vectors are replicable DNA constructs comprising synthetic or cDNA-derived DNA fragments encoding tumor-specific neoantigen peptides or bioequivalent analogs, operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcriptional unit generally comprises an assembly of (1) one or more gene elements having a regulatory role in gene expression, e.g., a transcription promoter or enhancer; (2) a structural or coding sequence transcribed to mRNA and translated to a protein; and (3) appropriate transcriptional and translational initiation and termination sequences, as further described herein. Such regulatory elements may include operator sequences that control transcription. Selective genes for promoting host replication ability (usually conferred by the origin of replication) and recognition of transformants may be further incorporated. DNA regions are operably linked when they are functionally related to one another. For example, the DNA of a signal peptide (secretion leader) is operably ligated to the polypeptide DNA if it is expressed as a precursor involved in polypeptide secretion; a promoter is operably ligated to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably ligated to a coding sequence if it is located in a position that enables translation. Generally, operably ligated means adjacent, and in the case of a secretion leader, it means adjacent and in the leading frame. Structural elements intended for use in yeast expression systems include leader sequences that enable extracellular secretion of translated proteins by the host cell. Alternatively, if a recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. In some cases, the final product may be obtained by subsequent cleavage of this residue from the expressed recombinant protein.

[0375] Examples of expression vectors useful for eukaryotic hosts, particularly mammals or humans, include vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids derived from Escherichia coli including pCR 1, pBR322, and pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and filamentous single-stranded DNA phages.

[0376] Suitable host cells for polypeptide expression include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli (E. coli) or Bacillus species. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (Pouwels). (See et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985)

[0377] Various mammalian or insect cell culture systems can also be used to express recombinant proteins, which is advantageous. Recombinant protein expression in mammalian cells is possible because such proteins are generally correctly folded, appropriately modified, and fully functional. Suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines with the ability to express appropriate vectors, such as L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may include non-transcription elements, such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed, as well as other 5' or 3' flanking non-transcription sequences and 5' or 3' untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor / receptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells have been reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0378] Proteins produced by transformed hosts can be purified by any preferred method. Such standard methods include chromatography (e.g., ion exchange, affinity and size exclusion column chromatography), centrifugation, solubility differential chromatography, or any other standard protein purification technique. Attaching affinity tags, such as hexahistidine, maltose-binding domains, influenza coat sequences, or glutathione-S-transferase, to the protein may allow for easy purification by passing it through a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0379] For example, the supernatant from a system that secretes recombinant protein into culture medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be added to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups, can be used. The matrix may be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the cancer stem cell protein-Fc composition can be further purified using one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having pendant methyl or other aliphatic groups. Some or all of the above purification steps can also be used in various combinations to provide a homogeneous recombinant protein.

[0380] Recombinant proteins produced in bacterial cultures can be isolated, for example, by first extracting them from a cell pellet, followed by one or more concentration, salting out, and performing a water-soluble ion exchange or size exclusion chromatography step. High-performance liquid chromatography (HPLC) may be used for the final purification step. Microbial cells used for recombinant protein expression can be destroyed by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysis agents.

[0381] In vivo peptide / polypeptide synthesis The present invention also intends to use nucleic acid molecules as a medium for delivering neoantigen peptides / polypeptides in vivo to targets that require them, for example, in the form of DNA / RNA vaccines (see, for example, International Publication No. 2012 / 159643 and International Publication No. 2012 / 159754, which are incorporated by reference in their entirety herein).

[0382] In one embodiment, the neoantigen is administered to a patient who needs it using a plasmid. These can be administered. These are plasmids that typically consist of a potent viral promoter and drive in vivo transcription and translation of the target gene (or complementary DNA) (Mor, et al., (1995). The Journal of Immunology 155(4):2039-2046). Sometimes, intron A may be included to improve mRNA stability and, consequently, increase protein expression (Leitner et al., (1997). The Journal of Immunology 159(12):6112-6119). Plasmids also contain potent polyadenylation / transcription termination signals such as bovine growth hormone or rabbit β-globulin polyadenylation sequences (Alarcon et al.,(1999).Adv.Parasitol.Advances in Parasitology 42:343-410; Robinson et al., (2000). Adv. Virus Res. Advances in Virus Research 55:1-74; Boehmet al., (1996). Journal of Immunological Methods 193(1):29-40). Sometimes, multicistron vectors are constructed to express two or more immunogens, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88).

[0383] Since plasmids are the "vegetation" from which immunogens are expressed, optimizing vector design to maximize protein expression is essential (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88). One way to enhance protein expression is to optimize the codon usage frequency of pathogenic mRNA for eukaryotic cells. Another consideration is the selection of promoters. Such promoters may be the SV40 promoter or Roussarcoma virus (RSV).

[0384] Plasmids can be introduced into animal tissues by several different methods. The two most common methods are injection of DNA in saline using a standard subcutaneous needle and gene gun delivery. A basic outline of the construction of DNA vaccine plasmids and their subsequent delivery to the host by these two methods is published in Scientific American (Weiner). As shown in et al., (1999) Scientific American 281(1):34-41), injection in saline is usually performed intramuscularly (IM) or intradermally (ID) in skeletal muscle, and DNA is delivered into the extracellular space. This can be done by electroporation, by transiently damaging muscle fibers with a myotoxin such as bupivacaine, or by using a hypertonic solution of saline or sucrose (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410). The immune response to this delivery method can be influenced by many factors, including needle type, needle positioning, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the animal receiving the injection (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410).

[0385] Another commonly used delivery method, gene gun delivery, involves ballistically accelerating plasmid DNA (pDNA) adsorbed onto gold or tungsten microparticles using compressed helium as an accelerator, and then delivering it into target cells (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88).

[0386] Alternative delivery methods include aerosol administration of naked DNA to mucosal surfaces such as the nasal and pulmonary mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88), and topical administration of pDNA to the ocular and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54:129-88). Examples include Press)54:129-88). Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA can also be delivered to cells after mild mechanical disruption of the cell membrane that makes the cell permeable. Such mild mechanical disruption of the membrane can be achieved by gently pushing the cell through a small opening ("Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells"). Macromolecules to Immune Cells)”,Sharei et al,PLOS ONE|DOI:10.1371 / journal.pone.0118803 April 13,2015).

[0387] The amount of DNA required to produce an effective immune response depends on the delivery method. Saline injection requires various amounts of DNA, ranging from 10 μg to 1 mg, while gene gun delivery requires only 1 / 100 to 1 / 1000th the amount of DNA needed for intramuscular saline injection to produce an effective immune response. Generally, 0.2 μg to 20 μg is required, although amounts as small as 16 ng have also been reported. These amounts vary by species; for example, mice require about one-tenth the amount of DNA required by primates. In saline injection, DNA is delivered to the extracellular space of the target tissue (usually muscle), where it must overcome physical barriers (to name a few, such as the basement membrane and a large amount of connective tissue) before being taken up by the cell, thus requiring more DNA. In contrast, gene gun delivery injects DNA directly into the cell, thus reducing "waste" (e.g., Sedegah et al.,(1994). Proceedings of the National Academy of Sciences of the United States of America 91(21):9866-9870; Daheshia et al.,(1997). The Journal of Immunology 159(4):1945-1952; Chen et al.,(1998). The Journal of Immunology 160(5):2425-2432; Sizemore(1995) Science 270(5234):299-302; Fynan et al. See al., (1993) Proc. Natl. Acad. Sci. USA 90(24):11478-82.

[0388] In one embodiment, the neobiotic vaccine or immunogenic composition may comprise a separate DNA plasmid encoding one or more neoantigen peptides / polypeptides, as identified, for example, according to the present invention. As discussed herein, the precise selection of the expression vector can depend on the peptide / polypeptide to be expressed and is well within the scope of the art. The expected persistence of the DNA construct (e.g., an episomal, non-replicating, non-integrated form in muscle cells) is expected to result in an increased duration of protection.

[0389] One or more neoantigen peptides of the present invention may be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system, an adeno-associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the neobiotic vaccine or immunogenic composition may include a virus-based vector used in a human patient in need of it, such as an adenovirus (e.g., Baden et al. "Recombinant adenovirus serotype 26 HIV-1 Env vaccine (IP)" First-in-human evaluation of the safety and immunogenicity of CAVD 001) immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine(IPCAVD See 001))''.J Infect Dis.2013 Jan 15;207(2):240-7 (incorporated herein by reference in whole). Plasmids that can be used for adeno-associated viruses, adenoviruses, and lentivirus delivery have been previously described (see, for example, U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent Application Publication No. 20080254008 (incorporated herein by reference)).

[0390] The peptides and polypeptides of the present invention can also be expressed using vectors, such as nucleic acid molecules considered herein, such as RNA or DNA plasmids, viral vectors, such as poxviruses, such as orthopoxvirus, avipoxvirus, or adenovirus, AAV, or lentivirus. This method involves the use of a vector to express the nucleotide sequence encoding the peptides of the present invention. When introduced into acutely or chronically infected or uninfected hosts, the vector expresses immunogenic peptides, thereby inducing a host CTL response.

[0391] Among the vectors that can be used in carrying out the present invention, retroviral gene transfer methods can be integrated into the host genome of cells and, in many cases, result in long-term expression of the inserted trans gene. In preferred embodiments, the retrovirus is a lentivirus. In addition, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of the retrovirus can be altered by introducing exogenous envelope proteins, thereby expanding the potential target population of target cells. Retroviruses can also be manipulated to enable conditional expression of the inserted trans gene, thus allowing only specific cell types to be infected with the lentivirus. Expression in specific cell types can be targeted using cell type-specific promoters. Lentiviral vectors are retroviral vectors (and thus both lentiviral vectors and retroviral vectors can be used in carrying out the present invention). Furthermore, lentiviral vectors are preferred because they can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the selection of retroviral gene transfer systems may depend on the target tissue. Retroviral vectors contain a cis-acting long-terminal repeat sequence with the ability to package exogenous sequences up to 6-10 kb. A minimal cis-acting LTR is sufficient for vector replication and packaging, and this is then used to incorporate the desired nucleic acid into target cells, resulting in persistent expression.Widely used retroviral vectors that can be used in carrying out the present invention include those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., (1992) J.Virol.66:2731-2739; Johann et al., (1992) J.Virol.66:1635-1640; Sommnerfelt et al., (1990) Virol.176:58-59; Wilson et al., (1998) J.Virol.63:2374-2378; Miller et al., (1991) J.Virol.65:2220-2224; and PCT / US94 / 05700).

[0392] Furthermore, in implementing the present invention, minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV), are also useful (e.g., Balagaan, (2006) J Gene Med; 8:275-285, online publication 21 November 2005 in Wiley InterSc). See ience (www.interscience.wiley.com).DOI:10.1002 / jgm.845. This vector may have a cytomegalovirus (CMV) promoter that drives the expression of a target gene. Accordingly, the present invention intends to include viral vectors, particularly retroviral vectors and lentiviral vectors, among vectors useful in carrying out the present invention.

[0393] Lentiviral vectors have been disclosed for the treatment of Parkinson's disease. See, for example, U.S. Patent Application Publication No. 20120295960, as well as U.S. Patent Nos. 7303910 and 7351585. Lentiviral vectors have also been disclosed with respect to delivery to the brain. See, for example, U.S. Patent Application Publication Nos. 20110293571; 20040013648, 20070025970, 20090111106, and U.S. Patent No. 7259015. In another embodiment, a lentiviral vector is used to deliver the vector to the brain of a subject being treated for a disease.

[0394] Regarding lentiviral vector systems useful in the practice of the present invention, reference is made to U.S. Patent Nos. 6428953, 6165782, 6013516, 5994136, 6312682, and 7,198,784, as well as the documents cited therein.

[0395] In one embodiment of the present specification, this delivery is by lentivirus. Zou et al. administered approximately 10 μl of recombinant lentivirus having a titer of 1 × 10 9 transducing units (TU) / ml by an intrathecal catheter. This type of dosage can be adapted or estimated for use of retroviruses or lentiviral vectors in the present invention. For transduction in tissues such as the brain, very small amounts are required, so the virus preparation is concentrated by ultracentrifugation. The resulting preparation should have at least 10 8 TU / ml, preferably 10 8 -~10 9 TU / ml, more preferably at least 10 9 TU / ml. Other concentration methods such as ultrafiltration or binding to a matrix and elution from the matrix may also be used.

[0396] In other embodiments, the amount of lentivirus administered is 1 × 10⁻¹⁶ as the total single dose for an average human weighing 75 kg, or as a total single dose adjusted according to the weight, size, and type of the subject. 5 Alternatively, approximately 1 x 10 5 Plaque-forming units (PFUs), 5 × 10 5 Or approximately 5 x 10 5 PFU, 1×10 6 Alternatively, approximately 1 x 10 6 PFU, 5x10 6 Or approximately 5 x 10 6 PFU, 1×10 7 Alternatively, approximately 1 x 10 7 PFU, 5x10 7 Or approximately 5 x 10 7 PFU, 1×10 8 Alternatively, approximately 1 x 10 8 PFU, 5x10 8 Or approximately 5 x 10 8 PFU, 1×10 9 Alternatively, approximately 1 x 10 9 PFU, 5x10 9 Or approximately 5 x 10 9 PFU, 1×10 10 Alternatively, approximately 1 x 10 10 PFU or 5×10 10 Or approximately 5 x 10 10 It can be a PFU. A person skilled in the art can determine the appropriate dosage. In the case of a virus, the appropriate dosage can be determined empirically.

[0397] Adenovirus vectors are also useful in carrying out the present invention. One advantage is that recombinant adenoviruses can efficiently transfer and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of the transferred nucleic acids. Furthermore, their ability to productively infect quiescent cells expands the usefulness of recombinant adenovirus vectors. In addition, they ensure the expression of nucleic acid products at high expression levels, sufficient to produce an immune response (see, for example, U.S. Patent No. 7,029,848, incorporated herein by reference).

[0398] Regarding adenovirus vectors useful in carrying out the present invention, U.S. Patent No. 6,95 Specification No. 5,808 is mentioned. The adenovirus vector used can be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome is publicly available (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) "The Sequence of the Genome of Adenovirus 5 and Comparison thereof with the Genome of Adenovirus 2"). "Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2"), Virology 186, 280-285 (the contents of which are incorporated herein by reference). The Ad35 vector is described in U.S. Patent Nos. 6,974,695, 6,913,922 and 6,869,794. The Ad11 vector is described in U.S. Patent No. 6,913,922. The C6 adenovirus vector is described in U.S. Patent Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975. The C7 vector is described in U.S. Patent No. 6,277,558. Adenovirus vectors that are E1-deficient or deleted, E3-deficient or deleted, and / or E4-deficient or deleted may also be used. Certain adenoviruses with mutations in the E1 region have improved safety margins because E1-deficient adenovirus mutants are replication-deficient or at least highly attenuated in non-permissible cells. Adenoviruses with mutations in the E3 region may have enhanced immunogenicity due to disruption of the mechanism by which adenoviruses downregulate MHC class I molecules. Adenoviruses with E4 mutations may have lower immunogenicity of adenovirus vectors due to suppression of late gene expression. Such vectors can be particularly useful when repeated revaccination using the same vector is desired. Adenovirus vectors with E1, E3, E4, E1 and E3, and E1 and E4 deletions or mutations may be used in accordance with the present invention. Furthermore, "gutless" adenovirus vectors, in which all viral genes are deleted, can also be used in accordance with the present invention.Such vectors require a helper virus for replication and a specific human 293 cell line expressing both E1a and Cre, conditions not found in the natural environment. Such "gutless" vectors are non-immunogenic and can therefore be administered multiple times for revaccination. "Gutless" adenovirus vectors can be used for the insertion of heterologous inserts / genes, such as the trans gene of the present invention, and can also be used for the co-delivery of multiple heterologous inserts / genes.

[0399] In one embodiment of this specification, delivery is via adenovirus, and the adenovirus is at least 1 × 10 5 It may be a single booster dose containing a particle (particle unit, also referred to as pu) adenovirus vector. In some embodiments herein, the dose is preferably at least about 1 × 10⁻⁶ 6 Particles (for example, about 1 × 10⁻⁶) 6 ~1 × 10 12 Particles), more preferably at least about 1 × 10 7 Particles, more preferably at least about 1 × 10⁻¹⁶ 8 Particles (for example, about 1 × 10⁻⁶) 8 ~1 × 10 11 Particles or approximately 1 × 10⁻⁶ 8 ~1 × 10 12 Particles), and most preferably at least about 1 × 10 9 Particles (for example, about 1 × 10⁻⁶) 9 ~1 × 10 10 Particles or approximately 1 × 10⁻⁶ 9 ~1 × 10 12 Particles), or even at least about 1 × 10⁻¹⁶ 10 Particles (for example, about 1 × 10⁻⁶) 10 ~1 × 10 12 It is an adenovirus vector (particle). Alternatively, the dose is approximately 1 × 10⁻⁶. 14 Less than a particle, preferably about 1 × 10⁻¹⁶ 13 Less than a particle, and more preferably about 1 × 10⁻¹⁶ 12 Less than a particle, and more preferably about 1 × 10⁻¹⁶ 11 Particles or smaller, and most preferably about 1 × 10⁻¹⁶ 10Smaller than a particle (for example, about 1 × 10⁻⁶) 9 It contains particles (articles or less). Therefore, the dose is, for example, about 1 × 10⁻⁶. 6 Particle unit (pu), approximately 2 x 10⁻⁶ 6 pu, approx. 4×10 6 pu, about 1×10 7 pu, approx. 2×10 7 pu, approx. 4×10 7 pu, about 1×10 8 pu, approx. 2×10 8 pu, approx. 4×10 8 pu, about 1×10 9 pu, approx. 2× 10 9 pu, approx. 4×10 9 pu, about 1×10 10 pu, approx. 2×10 10 pu, approx. 4×10 10 pu, about 1×10 11 pu, approx. 2×10 11 pu, approx. 4×10 11 pu, about 1×10 12 pu, approx. 2×10 12 PU, or approximately 4 x 10 12 This may include a single-dose adenovirus vector having a pu adenovirus vector. See, for example, the adenovirus vector of U.S. Patent No. 8,454,972 B2 granted to Nabel, et al. on 4 June 2013 (incorporated herein by reference), and the dosages in column 29, lines 36-58. In some embodiments herein, the adenovirus is delivered in multiple doses.

[0400] From an in vivo delivery perspective, AAV is advantageous compared to other viral vectors because it is less toxic due to not being integrated into the host genome and less likely to induce insertional mutagenesis. AAV has a packaging limit of 4.5 or 4.75 Kb. Viral production decreases significantly if the construct is larger than 4.5 or 4.75 Kb. Numerous promoters can be used to drive nucleic acid molecule expression. AAV ITR can act as a promoter and is advantageous because it does not require additional promoter elements. For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For expression in the brain, the following promoters can be used: synapsin I for any neuron, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Pol III promoters such as U6 or H1 can be used to drive RNA synthesis. Pol II promoters and intron cassettes can be used for guide RNA (gRNA) expression.

[0401] Regarding AAV vectors useful in carrying out the present invention, U.S. Patent Nos. 5,658,785, 7,115,391, 7172,893, 6953,690, 6936,466, 6924,128, 6893,865, 6793,926, 6537,540, 6475,769, and 6258,595, as well as the documents cited in these specifications, are referenced.

[0402] With respect to AAV, the AAV may be AAV1, AAV2, AAV5, or any combination thereof. The AAV can be selected in relation to the target cells; for example, for targeting brain or nerve cells, AAV serotype 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected; and for targeting heart tissue, AAV4 can be selected. AAV8 is useful for delivery to the liver. The above promoters and vectors are individually preferred.

[0403] In certain embodiments herein, delivery is via AAV. A therapeutically effective dosage for in vivo delivery of AAV to humans is about 1×10 10 ~ about 1×10 50 functional AAV / ml solution, which is considered to be in the range of about 20 to about 50 ml of saline. The dosage can be adjusted to balance the therapeutic benefit and any side effects. In certain embodiments herein, the AAV dosage is generally about 1×10 5 ~ 1×10 50 genomic AAV, about 1×10 8 ~ 1×10 20 genomic AAV, about 1×10 10 ~ about 1×10 16 genome, or about 1×10 11 ~ about 1×10 16 in the concentration range of genomic AAV. The human dosage can be about 1×10 13 genomic AAV. Such concentration can be delivered in a carrier solution of about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml. In a preferred embodiment, AAV with a titer of about 2×10 13 virus genomes / milliliter is used, and each of the striatal hemispheres of the mouse receives a single 500 nanoliter injection. Other effective dosages can be readily established by routine tests of creating a dose-response curve by those skilled in the art . See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., column 27, lines 45 - 60, issued March 26, 2013.

[0404] In another embodiment, effective activation of a cellular immune response to a neomicrobial vaccine or immunogenic composition can be achieved by the expression of relevant neoantigens in the vaccine or immunogenic composition in non-pathogenic microorganisms. Well-known examples of such microorganisms include Mycobacterium bovis (BCG), Salmonella, and Pseudomona (see U.S. Patent No. 6,991,797, which is incorporated herein by reference in its entirety).

[0405] In another embodiment, poxviruses are used in neoplastic vaccines or immunogenic compositions. Poxviruses include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (e.g., Verardiet al., Hum Vaccin Immunother. 2012). See Jul;8(7):961-70; and Moss, Vaccine. 2013;31(39):4220-4222). Poxvirus expression vectors were reported in 1982 and quickly became widely used in vaccine development and research in numerous fields. The advantages of this vector include its simple construction, ability to accommodate large amounts of foreign DNA, and high expression levels.

[0406] Information regarding poxviruses that can be used in carrying out the present invention, such as, in particular, Chordopoxvirinae poxviruses (vertebrate poxviruses), such as orthopoxvirus and avipoxvirus, such as vaccinia virus (e.g., Weiss strain, WR strain (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 strain, ALVAC), fowlpox virus (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoonpox, their synthetic or naturally occurring recombinants, their use, and methods for producing and using such recombinants can be found in scientific literature and patent documents such as the following: • U.S. Patent Nos. 4,603,112, 4,769,330, 5,110,587, 5,174,993, 5,364,773, 5,762,938, 5,494,807, 5,766,597, 7,767,449, 6,780,407, 6,537,594, 6,265,1 Specification No. 89, Specification No. 6,214,353, Specification No. 6,130,066, Specification No. 6,004,777, Specification No. 5,990,091, Specification No. 5,942,235, Specification No. 5,833, Specification No. 975,5, Specification No. 766,597, Specification No. 5,756,101, Specification No. 7,045,313, Specification No. 6,780,417, Specification No. 8,470,598, Specification No. 8,372 , 622, 8,268,329, 8,268,325, 8,236,560, 8,163,293, 7,964,398, 7,96 Specification No. 4,396, Specification No. 7,964,395, Specification No. 7,939,086, Specification No. 7,923,017, Specification No. 7,897,156, Specification No. 7,892,533, Specification No. 7,6 Specification No. 28,980, Specification No. 7,459,270, Specification No. 7,445,924, Specification No. 7,384,644, Specification No. 7,335,364, Specification No. 7,189,536, Specification No. 7,097,842, Specification No. 6,913,752, Specification No. 6,761,893, Specification No. 6,682,743, Specification No. 5,770,212, Specification No. 5,766,882, and the same Specification No. 5,989,562, and ·Panicali,D.Proc.Natl.Acad.Sci.1982;79;4927-493,Panicali D.Proc.Natl.Acad.Sci.1983;80(17):5364-8,Mackett,M.Proc.Natl.Acad.Sci.1982;79:7415-7419,Smith GL.Proc.Natl.Acad.Sci.1983;80(23):7155-9,Smith GL.Nature 1983;302:490-5,Sullivan VJ.Gen.Vir.1987;68:2587-98,Perkus M Journal of Leukocyte Biology 1995;58:1-13,Yilma TD.Vaccine 1989;7:484-485,Brochier B.Nature 1991;354:520-22,Wiktor,TJ.Proc.Natl Acd.Sci.1984;81:7194-8,Rupprecht,CE.Proc.Natl Acd.Sci.1986;83:7947-50,Poulet,H Vaccine 2007;25(Jul):5606-12,Weyer J.Vaccine 2009;27(Nov):7198-201,Buller,RM Nature 1985;317(6040):813-5,Buller RM.J.Virol.1988;62(3):866-74,Flexner,C.Nature 1987;330(6145):259-62,Shida,H.J.Virol.1988;62(12):4474-80,Kotwal,GJ.J.Virol.1989;63(2):600-6,Child,SJ.Virology 1990;174(2):625-9,Mayr A.Zentralbl Bakteriol 1978;167(5,6):375-9,Antoine G.Virology.1998;244(2):365-96,Wyatt,LS.Virology 1998;251(2):334-42,Sancho,MC.J.Virol.2002;76(16);8313-34,Gallego-Gomez,JC.J.Virol.2003;77(19);10606-22),Goebel SJ.Virology 1990;(a,b)179:247-66,Tartaglia,J.Virol.1992;188(1):217-32,Najera JL.J.Virol.2006;80(12):6033-47,Najera,JL.J.Virol.2006;80:6033-6047,Gomez,CE.J.Gen.Virol.2007;88:2473-78,Mooij.Of,P.Jour. Virol.2008;82:2975-2988,Gomez,CE.Curr.Gene Ther.2011;11:189-217,Cox,W.Virology 1993;195:845-50,Perkus,M.Jour.Of.Leukocyte-15 TJ3;5 Gen Virology 1998;79(5):1159-67,Amara R.Science 2001;292:69-74,Hel,Z.,J.Immunol.2001;167:7180-9,Gherardi MM.J.Virol.20704,Adi.7-Vaccine 2004;22:3395-3403,Bissht H.Proc.Nat.Aca.Sci.2004;101:6641-46,McCurdy LH.Clin.Inf.Dis 2004;38:1749-53,Earl PL:18,Chen-4 2004 ZJVirol.2005;79:2678-2688,Najera JL.J.Virol.2006;80(12):6033-47,Nam JH.Acta.Virol.2007;51:125-30,Antonis AF.B.B71;8-4:2007 J.Vaccine 2007;25:4213-22,Ferrier-Rembert A.Kaufcine 2008;26(14):1794-804,Corbett M.Proc.Natl.Acad.Sci.2008;105(6):2046-51, HL.,J.Clin.Oncol.2004;22:2122-32,Amato,RJ.Clin.Cancer Res.2008. ;14(22):7504-10,Dreicer R.Invest New Drugs 2009;27(4):379-86,Kantoff PW.J.Clin.Oncol.2010,28,1099-1105,Amato RJ.J.Clin.Can.Res.2010;16(22):5539-47,Kim,DW.Hum.Vaccine.2010;6:784-791,Ouard,S.Cancer Immunol.Immunother.2011;60:261-71,Wyatt,LS.Aids. Res.Hum.Retroviruses.2004;20:645-53,Gomez,CE.Virus Research 2004;105:11-22,Webster,DP.Proc.Natl.Acad.Sci.2005;102:4836-4,Huang,X.Vaccine 2007;25:8874-84,Gomez,CE.Vaccine 2007a;25:2863-85,Esteban M.Hum.Vaccine 2009;5:867-871,Gomez,CE.Curr.Gene therapy 2008;8(2):97-97-Plos One,WT.K. 2009;4(6):5934,Scriba,TJ.Eur.Jour.Immuno.2010;40(1):279-90,Corbett,M.Proc. Natl.Acad.Sci.2008;105:2046-2051,Midgley,CM.J.Gen.Virol.2008;89:2992-97,Von Krempelhuber,A.Vaccine 2010;28:1209-16,Perreau,MJOf Virol.2011;Oct:9854-62,Pantaleo,G.Curr Opin HIV-AIDS.2010;5:391-396 (Sunshine, Swedish Beauty)

[0407] In another embodiment, vaccinia virus is used in a neoplastic vaccine or immunogenic composition to express a neoantigen (Rolph et al., "Recombinant viruses as vaccines and immunological tools," Curr Opin Immunol 9:517-524, 1997). Recombinant vaccinia virus has the ability to replicate in the cytoplasm of infected host cells, and thus the target polypeptide can induce an immune response. Furthermore, poxviruses are widely used as vaccine or immunogenic composition vectors because they can target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, particularly antigen-presenting cells, and because of their self-adjuvant ability.

[0408] In another embodiment, ALVAC is used as a vector in a neoplasm vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express an exogenous trans gene and is used as a method of vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. "Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule" Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang KY, et al. "Pilot study of a dual gene recombinant Avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 trans gene in patients with recurrent CEA-expressing adenocarcinoma" recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B 7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas). Clin Cancer Res 2000;6:2219-28; Musey L, Ding Y, Elizaga M, et al. "HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1 uninfected individuals." T cell immunity in HIV-1-uninfected individuals). J Immunol 2003;171:1094-101; Paoletti E. "Applications of pox virus vectors to vaccination: an update". Proc Natl Acad Sci USA 1996;93:11349-53; U.S. Patent No. 7,255,862). In a Phase I clinical trial, ALVAC viruses expressing the tumor antigen CEA demonstrated a superior safety profile and resulted in an increased CEA-specific T cell response in selected patients; however, no objective clinical response was observed (Marshall JL, Hawkins MJ, Tsang KY, et al. "Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen". J Clin Oncol 1999;17:332-7).

[0409] In another embodiment, modified vaccinia ankara (MVA) virus can be used as a viral vector for neoantigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and has been created by serially passage the Ankara strain of vaccinia virus (CVA) for approximately 570 generations in chicken embryonic fibroblasts (see Mayer, A., et al., Infection 3, 6-14, 1975 for a review). As a result of such passage, the resulting MVA virus contains 31 kilobases less genomic information compared to CVA and is highly host cell-restrictive (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, i.e., reduced virulence or infectivity, but still retains excellent immunogenicity. When tested in various animal models, MVA has been proven nontoxic even in immunosuppressed individuals. Furthermore, MVA-BN(registered trademark)-HER2 is an immunotherapy candidate designed for the treatment of HER-2-positive breast cancer and is currently in clinical trials (Mandl et al., Cancer Immunol Immunother. Jan 2012;61(1):19-29). Methods for preparing and using recombinant MVA are described (see, for example, U.S. Patent Nos. 8,309,098 and 5,185,146, which are incorporated herein by reference).

[0410] In another embodiment, modified vaccinia virus Copenhagen strains, NYVAC, and NYVAC variants are used as vectors (see U.S. Patent No. 7,255,862; International Publication No. 95 / 30018; U.S. Patent Nos. 5,364,773 and 5,494,807 (referred to herein by reference in their entirety)).

[0411] In one embodiment, recombinant virus particles of a vaccine or immunogenic composition are administered to a patient who needs it. The dosage of the neoantigen expressed can be in the range of several micrograms to several hundred micrograms, for example, 5 - 500 μg. The vaccine or immunogenic composition can be administered in any amount suitable to achieve expression at such dosage levels. The virus particles can be administered to a patient who needs it in an amount of at least about 10 pfu, or can be transfected into cells; thus, the virus particles are preferably at least about 10 3.5 pfu to about 10 4 pfu and are administered to a patient who needs it, or infect cells or are transfected into cells; however, at least about 10 6 pfu can be administered to a patient who needs it, and a more preferred dosage can be at least about 10 8 pfu to about 10 7 [[ID= ())]]pfu and can be about 10 9 pfu. The dosage for NYVAC is applicable to ALVAC, MVA, MVA - BN, and avipox, such as canarypox and fowlpox.

[0412] Vaccine or immunogenic composition adjuvant An effective vaccine or immunogenic composition advantageously contains a potent adjuvant to elicit an immune response. As described herein, poly-ICLC, which is a TLR3 agonist and the RNA helicase domains of MDA5 and RIG3, exhibits several desirable properties for vaccine or immunogenic composition adjuvants. These properties include induction of local and systemic activation of immune cells in vivo, production of stimulating chemokines and cytokines, and stimulation of antigen presentation by DCs. Furthermore, poly-ICLC can induce a sustained CD4+ and CD8+ response in humans. Importantly, significant similarities in the upregulation of transcriptional and signaling pathways were observed between subjects vaccinated with poly-ICLC and volunteers who had previously received a highly effective replication-competent yellow fever vaccine. In addition, in a recent Phase 1 study, more than 90% of ovarian cancer patients immunized with poly-ICLC in combination with the NY-ESO-1 peptide vaccine (in addition to Montanide) showed induction of CD4+ and CD8+ T cells and an antibody response to the peptide. Simultaneously, poly-ICLC has been extensively tested in more than 25 clinical trials to date, exhibiting a relatively safe toxicity profile. In addition to being potent and specific immunogens, neoantigen peptides can be used in combination with adjuvants (e.g., poly-ICLC) or other antineoplastic agents. While not constrained by theory, these neoantigens are expected to evade central thymic tolerance (thus enabling a more potent antitumor T cell response) while reducing the potential for autoimmunity (e.g., by avoiding targeting of normal autoantigens). An effective immune response favorably involves a potent adjuvant to activate the immune system (Speiser and Romero, "Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity," Seminars in Immunol 22:144 (2010)).For example, Toll-like receptors (TLRs) have emerged as powerful sensors of microbial and viral pathogen "danger signals" that effectively induce the innate immune system and then the adaptive immune system (Bhardwaj and Gnjatic, "TLR AGONISTS: Are They Good Adjuvants?" Cancer J.16:382-391 (2010)). Among TLR agonists, poly-ICLC (synthetic double-stranded RNA mimetic) is one of the most potent activators of bone marrow-derived dendritic cells. In human volunteer trials, poly-ICLC has been shown to be safe and induce a gene expression profile in peripheral blood cells that is comparable to that induced by YF-17D, one of the most potent attenuated live viral vaccines (Caskey et al, "Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans," J Exp Med 208:2357 (2011)). In a preferred embodiment, Hiltonol®, a GMP formulation of poly-ICLC prepared by Oncovir, Inc., acts as an adjuvant. It is used as follows. In other embodiments, other adjuvants described herein are envisioned. For example, oil in water, water in oil, or multiphase W / O / W; see, for example, U.S. Patent No. 7,608,279 and Aucouturier et al, Vaccine 19 (2001), 2666-2672, and their references.

[0413] Adaptation Examples of cancers and cancerous conditions that can be treated by the treatments described herein include, but are not limited to, patients who have been diagnosed with cancer or are at risk of developing cancer and who require such treatment. The subjects include solid tumors, such as breast, ovarian, prostate, lung, kidney, stomach, colon, testis, head and neck, pancreas, brain, melanoma, and other tissue organ tumors; hematological malignancies, such as lymphomas and leukemias including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma; tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other CNS sites, e.g., glioblastoma or medulloblastoma); head and / or neck cancers; breast tumors; circulatory system tumors (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); and hematological and lymphatic system tumors (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-associated lymphoma, malignant immunoproliferative disease). Diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphocytic leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell types, leukemias of unspecified cell types, unspecified malignant neoplasms of lymphoid tissue, hematopoietic tissue and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma); excretory system (e.g., kidneys, renal pelvis, urine) Tumors of the urinary tract (e.g., esophagus, stomach, small intestine, colon, colorectum, rectosigmoid junction, rectum, anus, and anal canal); tumors of the liver and intrahepatic bile ducts, gallbladder, and other biliary sites, pancreas, and other digestive organs; tumors of the oral cavity (e.g., lips, tongue, gums, floor of the mouth, palate, parotid gland, salivary gland, tonsils, oropharynx, nasopharynx, piriform fossa); Tumors of the sinus, hypopharynx, and other oral regions; tumors of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovaries, and other female reproductive organs, placenta, penis, prostate, testes, and other male reproductive organs); tumors of the airway (e.g., nasal cavity, middle ear, sinuses, larynx, trachea, bronchi, and lungs, e.g., small cell lung cancer and non-small cell lung cancer); tumors of the skeletal system (e.g., limbs, articular cartilage, and bones and articular cartilage of other regions); tumors of the skin (e.g., malignant melanoma, non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, mesothelioma, Kaposi's sarcoma);The subjects may also have tumors involving other tissues, including peripheral nerves and the autonomic nervous system, connective tissue and soft tissue, retroperitoneum and peritoneum, the eyes, thyroid gland, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites. Therefore, the subject population described herein may be affected by one of the above types of cancer. In other embodiments, the subject population may consist entirely of subjects with solid tumors, or entirely of subjects with humoral tumors.

[0414] In particular, the treatment of non-Hodgkin lymphoma (NHL), clear cell renal carcinoma (ccRCC), metastatic melanoma, sarcoma, leukemia or bladder cancer, colon cancer, brain cancer, breast cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, pancreatic cancer or prostate cancer is of interest. In certain embodiments, melanoma is a high-risk melanoma.

[0415] Cancers that can be treated using the therapies described herein may, in particular, include cases that are refractory to treatment with other chemotherapeutic agents. The term “refractory” as used herein refers to cancers (and / or metastases thereof) that show no or very weak antiproliferative response after treatment with another chemotherapeutic agent (e.g., no or very weak inhibition of tumor growth). These are cancers that cannot be adequately treated with other chemotherapeutic agents. Refractory cancers include (i) cancers that have already failed one or more chemotherapeutic agents in the patient's treatment, as well as (ii) cancers that are refractory by other means, such as biopsy and culture in the presence of chemotherapeutic agents. Cancers that may be shown to be cancerous are also included.

[0416] The treatments described herein are also applicable to patients who require such treatment and who have not received such treatment before.

[0417] The treatments described herein are also applicable to cases where no neoplasms are detected in the subject but the risk of disease recurrence is high.

[0418] Furthermore, the treatment of patients who have undergone or require autologous hematopoietic stem cell transplantation (AHSCT), and more specifically, patients exhibiting residual disease after AHSCT, is of particular interest. The post-AHSCT situation is characterized by a small amount of residual disease, infusion of immune cells against a homeostatic proliferative state, and the absence of any standard treatment to delay relapse. These characteristics present a unique opportunity to delay disease relapse using the claimed neoplasm vaccine or immunogenic composition.

[0419] Pharmaceutical composition / delivery method The present invention also relates to pharmaceutical compositions comprising an effective amount of one or more neoantigen peptides (including pharmaceutically acceptable salts thereof) as described herein, in combination with optionally pharmaceutically acceptable carriers, excipients, or additives.

[0420] When administered in combination, the therapeutic agent (i.e., the neoantigen peptide) may be formulated as separate compositions administered at the same or different time points, or the therapeutic agent may be administered as a single composition.

[0421] This composition may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once a year. The dosing interval can be adjusted according to the individual patient's needs. For longer dosing intervals, sustained-release formulations or depot formulations may be used.

[0422] The compositions of the present invention can be used to treat acute diseases and disease conditions, and can also be used to treat chronic conditions. More specifically, the compositions of the present invention are used in methods for treating or preventing neoplasms. In certain embodiments, the compounds of the present invention are administered over periods exceeding 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, 10 years, or 15 years; or over any period range in days, months, or years (e.g., 4 weeks to 15 years, 6 months to 20 years) where the lower end of the range is any period between 14 days and 15 years and the upper end of the range is between 15 days and 20 years. In some cases, it may be advantageous to administer the compounds of the present invention over the lifetime of the patient. In preferred embodiments, the patient is monitored to check for progression of the disease or disability, and the dose is adjusted accordingly. In a preferred embodiment, the treatment according to the present invention is effective for at least two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, fifteen years, twenty years, or for the lifetime of the subject.

[0423] Surgical resection involves surgically removing abnormal cancerous tissue such as mediastinal tumors, neurogenic tumors, germ cell tumors, or thymomas. In certain embodiments, administration of the composition is initiated after tumor resection. In other embodiments, administration of the neoplasm vaccine or immunogenic composition is initiated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 weeks or later after tumor resection. Preferably, administration of the neoplasm vaccine or immunogenic composition is initiated 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after tumor resection.

[0424] A prime / boost regimen refers to the sequential administration of a vaccine or immunogenic or immunological composition. In certain embodiments, the administration of a neoplasm vaccine or immunogenic composition constitutes a prime / boost dosing regimen, for example, with the administration of the neoplasm vaccine or immunogenic composition as priming in weeks 1, 2, 3, or 4, and the administration of the neoplasm vaccine or immunogenic composition as boosting in months 2, 3, or 4. In another embodiment, a higher cytotoxic T cell response is induced using a heterologous prime-boost strategy (see Schneider et al., "Induction of CD8+ T cells using heterologous prime-boost immunisation strategies," Immunological Reviews Volume 170, Issue 1, pages 29-38, August 1999). In yet another embodiment, a protein boost follows priming using DNA encoding a neoantigen. In another embodiment, priming with a protein is followed by a boost with a virus encoding a neoantigen. In yet another embodiment, priming is performed using a virus encoding a neoantigen, and then a boost is performed using a different virus. In yet another embodiment, priming is performed using a protein, and then a boost is performed using DNA. In a preferred embodiment, a T cell response is primed with a DNA vaccine or immunogenic composition, and that response is boosted with a recombinant viral vaccine or immunogenic composition. In yet another preferred embodiment, a viral vaccine or immunogenic composition is co-administered with a protein or DNA vaccine or immunogenic composition and acts as an adjuvant for the protein or DNA vaccine or immunogenic composition.Next, the patient may be boosted with either a viral vaccine or immunogenic composition, a protein, or a DNA vaccine or immunogenic composition (Hutchings et al., "Combination of protein and viral vaccines induces potent cellular and humoral immune response and enhanced protection against mouse malaria attack infection"). "Responses and enhanced protection from murine malaria challenge." Infect Immun. 2007 Dec;75(12):5819-26. See Epub 2007 Oct 1.

[0425] The pharmaceutical composition can be processed according to conventional pharmaceutical methods for preparing pharmaceutical agents for administration to patients in need, including humans and other mammals.

[0426] Modification of neoantigen peptides can affect the peptide's solubility, bioavailability, and metabolic rate, and thus may lead to control over the delivery of the active species. Solubility can be evaluated by preparing the neoantigen peptide and testing it using known methods that are well within the scope of the conventional techniques of those skilled in the art.

[0427] In certain embodiments of this pharmaceutical composition, the pharmaceutically acceptable carrier comprises water. In certain embodiments, the pharmaceutically acceptable carrier further comprises dextrose. In certain embodiments, the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide. In certain embodiments, this pharmaceutical composition further comprises an immunomodulator or adjuvant. In certain embodiments, the immunomodulator or adjuvant is polyICLC, STING agonist, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, The immunomodulator or adjuvant is selected from the group consisting of MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, and Aquila's QS21 stimulon. In certain embodiments, the immunomodulator or adjuvant includes poly-ICLC.

[0428] Xanthenone derivatives, such as bazimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)), may also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives may also be administered in parallel with the vaccine or immunogenic composition of the present invention, for example, by systemic or intratumoral delivery, to stimulate immunity at the tumor site. Although not constrained by theory, it is thought that such xanthenone derivatives act by stimulating interferon (IFN) production via the IFN gene-stimulating factor (STING) receptor (see, for example, Conlon et al. (2013) "Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid," Journal of Immunology, 190:5216-25 and Kim et al. (2013) "Anticancer Flavonoids are Mouse-Selective STING Agonists," 8:1396-1401).

[0429] The vaccine or immunological composition may also include an adjuvant compound selected from acrylic or methacrylic polymers and maleic anhydride-alkenyl derivative copolymers. More specifically, it is a polymer (carbomer) in which acrylic acid or methacrylic acid is crosslinked with a polyalkenyl ether of a sugar or polyhydric alcohol, more specifically a polymer crosslinked with allyl clucrose or allyl pentaerythritol. It may also be a copolymer in which maleic anhydride and ethylene are crosslinked with, for example, divinyl ether (see U.S. Patent No. 6,713,068, which is incorporated herein by reference in whole).

[0430] In certain embodiments, the pH adjuster can stabilize the adjuvant or immunomodulator as described herein.

[0431] In certain embodiments, the pharmaceutical composition comprises one to five peptides, dimethyl sulfoxide (DMSO), dextrose, water, succinate, poly-I:poly-C, poly-L-lysine, carboxymethylcellulose, and chloride. In certain embodiments, each of the one to five peptides is present at a concentration of 300 μg / ml. In certain embodiments, the pharmaceutical composition contains ≤3 volume% DMSO. In certain embodiments, the pharmaceutical composition contains 3.6–3.7% dextrose in water. In certain embodiments, the pharmaceutical composition contains 3.6–3.7 mM succinate (e.g., sodium succinate) or a salt thereof. In certain embodiments, the pharmaceutical composition contains 0.5 mg / ml poly-I:poly-C. In certain embodiments, the pharmaceutical composition contains 0.375 mg / ml poly-L-lysine. In certain embodiments, the pharmaceutical composition contains 1.25 mg / ml sodium carboxymethylcellulose. In certain embodiments, the pharmaceutical composition contains 0.225% sodium chloride.

[0432] A pharmaceutical composition comprises a tumor-specific neoantigen peptide as described herein in a therapeutically effective amount for treating the diseases and conditions described herein (e.g., neoplasms / tumors), optionally combined with pharmaceutically acceptable additives, carriers, and / or excipients. Those skilled in the art will recognize from this disclosure and the knowledge in the art that the therapeutically effective amount of one or more compounds according to the present invention may vary depending on the condition to be treated, its severity, the treatment regimen used, the pharmacokinetics of the drugs used, and the patient (animal or human) being treated.

[0433] To prepare a pharmaceutical composition according to the present invention, one or more therapeutically effective amounts of the compounds according to the present invention are preferably thoroughly mixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical formulation techniques so that a dose can be produced. The carrier can take a wide variety of forms depending on the preferred form of administration, such as ocular, oral, topical, or parenteral formulations, such as gels, creams, ointments, lotions, and time-release implantable formulations. When preparing a pharmaceutical composition as an oral dosage form, any ordinary pharmaceutical medium can be used. Accordingly, suitable carriers and additives, including water, glycols, oils, alcohols, flavoring agents, preservatives, and colorants, can be used for liquid oral formulations such as suspensions, elixirs, and solutions. Suitable carriers and additives, including starch, sugar carriers, such as dextrose, mannitol, lactose, and related carriers, diluents, granulators, lubricants, binders, and disintegrants, can be used for solid oral formulations such as powders, tablets, and capsules, and for solid formulations such as suppositories. If necessary, the tablets or capsules may be enterically coated or sustained-release by standard techniques.

[0434] The active compound is contained in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to the patient a therapeutically effective dose for the desired symptoms without causing significant toxicity to the patient being treated.

[0435] Oral compositions generally contain an inert diluent or food carrier. Oral compositions may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative may be combined with excipients and used in the form of tablets, lozenges, or capsules. Drug-compatible binders and / or auxiliary materials may be included as part of the composition.

[0436] Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; dispersants such as alginic acid or corn starch; lubricants such as magnesium stearate; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. If the dosage unit form is a capsule, it may contain a liquid carrier such as fatty oil in addition to the materials described herein. In addition, the dosage unit form may contain various other materials that modify the physical form of the dosage unit, such as sugar, shellac, or enteric coating.

[0437] Formulations of the present invention suitable for oral administration may be provided as individual units, each containing a predetermined amount of the active ingredient, such as capsules, cachets, or tablets; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions and boluses.

[0438] Tablets may be manufactured by compression or molding, possibly with one or more auxiliary components. Compressed tablets may contain the active ingredient in a free-flowing form such as a powder or granules, possibly with a binder, lubricant, etc. It can be prepared by mixing with a lubricant, an inert diluent, a preservative, a surfactant, or a dispersant and compressing it in a suitable machine. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored and may be formulated to provide sustained or controlled release of the active ingredient.

[0439] Methods for formulating such sustained-release or controlled-release compositions of pharmaceutically active ingredients are known in the art and are described in several granted U.S. patents, including, but not limited to, U.S. Patent Nos. 3,870,790; Nos. 4,226,859; Nos. 4,369,172; Nos. 4,842,866 and Nos. 5,705,190 (these disclosures are incorporated herein by reference as a whole). Coatings can be used to deliver compounds to the intestines (see, for example, U.S. Patent Nos. 6,638,534, Nos. 5,541,171, Nos. 5,217,720 and Nos. 6,569,457 and the references therein).

[0440] The act...

Claims

1. A composition that is an immunogenic composition or vaccine composition for use in the treatment or prevention of chromophobe cell carcinoma of the kidney (KICH), the following: (a) Recombinant or synthetic neoantigen peptides that bind to HLA proteins; (b) Polynucleotides encoding recombinant neoantigen peptides; (c) Antigen-presenting cells (APCs) including (a) or (b); (d) T cells stimulated with an APC containing (a) or (b); or (e) T cell receptors (TCRs) that bind to MHC:peptide complexes containing tumor-specific neoepitopes of recombinant or synthetic neoantigen peptides that bind to HLA proteins. Includes, Neoantigen peptides contain tumor-specific neoepitopes of 8-10 amino acids in length, which include amino acid sequences encoded in the downstream sequence of a frameshift mutation in a gene. The gene in question is UBR5, and its neoantigen peptides are KNSPCCQKK (SEQ ID NO: 30664), KLRVQNQGH (SEQ ID NO: 30665), RVQNQGHLL (SEQ ID NO: 30666), VQNQGHHLLM (SEQ ID NO: 30667), LRVQNQGHL (SEQ ID NO: 30668), NQGHHLLMIL (SEQ ID NO: 30669), and MQNRQKKKGK (SEQ ID NO: 30670). The composition is a peptide comprising an amino acid sequence selected from the group consisting of KGKNSPCCQK (SEQ ID NO: 30671), KLRVQNQGHL (SEQ ID NO: 30672), RVQNQGHLLM (SEQ ID NO: 30673), LRVQNQGHLL (SEQ ID NO: 30674), VQNQGHLLMI (SEQ ID NO: 30675), and NQGHLLMILL (SEQ ID NO: 30676).

2. The composition according to claim 1, wherein the gene is UBR5 and the frameshift is the K2120fs mutation.

3. The composition according to claim 1, wherein the gene is UBR5 and contains the c.6360delA mutation.

4. The composition according to claim 1, wherein the tumor-specific neoepitope is (a) encoded by a gene of a human cancer cell, (b) expressed by a human cancer cell, (c) not expressed by a gene of a human non-cancer cell, and (d) not encoded by a gene of a non-cancer cell.

5. The composition according to claim 1, which is a vaccine.

6. The composition according to claim 1, wherein the polynucleotide encoding the recombinant neoantigen peptide is DNA.

7. The composition according to claim 1, wherein the polynucleotide encoding the recombinant neoantigen peptide is messenger RNA (mRNA).

8. A pharmaceutical composition for use in the treatment or prevention of KICH in a human subject as needed, comprising the composition according to claim 1, and a pharmaceutically acceptable carrier, excipient, or diluent.

9. The pharmaceutical composition according to claim 8, further comprising an adjuvant.

10. Use of the pharmaceutical composition according to claim 9 in the manufacture of a pharmaceutical for treating or preventing KICH in human subjects who require it.

11. The use according to claim 10, wherein the pharmaceutical composition is administered prophylactically.

12. The use according to claim 10, wherein the human subject is at risk of developing KICH.

13. The pharmaceutical composition according to claim 8, administered prophylactically.

14. The pharmaceutical composition according to claim 8, wherein human subjects are at risk of developing KICH.

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