Compositions and methods for targeting mutant ras

Abstract

The present invention relates to compositions and methods for treating cancers associated with mutant RAS. In certain aspects, the present invention relates to antigenic RAS peptide fragments and T cell receptors that bind to specific mutant RAS peptide fragments in the context of specific HLA types.

Description

[0001] CITATION OF RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 796,733, filed January 25, 2019, which is incorporated by reference in its entirety. BACKGROUND

[0003] Somatic mutations have been identified as a common driver of tumorigenesis. Activating point mutations in the Ras gene were the first somatic point mutations identified in human cancer. RAS mutations are the most common somatic mutations found in human cancer and they contribute to the pathogenesis of a variety of highly prevalent malignancies, including lung, colorectal, and pancreatic ductal adenocarcinomas. Mutant RAS is an attractive target for treating cancer because it is considered a driver mutation uniquely expressed by cancer cells and is important for tumor growth and survival. These mutations typically involve the codon 12 position of the RAS protein, and the amino acid changes are highly conserved, most commonly caused by G12C, G12D, G12R, and G12V amino acid substitutions. Pathological RAS mutations are functional gain-of-function mutations that result in constitutive activation of intracellular GTPase signaling that promotes cell growth. RAS mutations can be found at high frequencies in certain cancer types. For example, G12D and G12V mutations are present in 60-70% of pancreatic cancers and 20-30% of colorectal cancers. Unfortunately, there are no effective pharmacological inhibitors of RAS oncoproteins.

[0004] Accordingly, there is a need in the art for compositions and methods for treating mutant RAS-associated cancers. The present invention addresses and satisfies these and other needs. SUMMARY

[0005] In one aspect, the present invention provides immunogenic compositions comprising a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS. In one embodiment, the peptide comprises a G12C, G12D, G12R, or G12V mutation. In one embodiment, the mutant RAS peptide comprises 9 or 10 amino acid residues.

[0006] In one embodiment, the mutant RAS peptide comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16. In one embodiment, the mutant RAS peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16.

[0007] In one embodiment, the present application provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS.

[0008] In one embodiment, the present application provides a cell modified to comprise or express a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is selected from the group consisting of an antigen presenting cell, a B cell, a dendritic cell, a macrophage, a Langerhans cell, a T cell, a NK cell, a NK T cell.

[0009] In one aspect, the present application provides a method of inducing an immune response in a subject, the method comprising administering to the subject an immunological composition comprising a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS; or a nucleic acid molecule encoding a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS.

[0010] In one embodiment, the method comprises identifying the HLA type of the subject, and administering to the subject a composition comprising or encoding a mutant RAS peptide comprising a mutation at a position relative to G12 of wild-type RAS, wherein the mutant RAS peptide binds to the identified HLA molecule of the subject. In one embodiment, the subject has or is at risk of having a RAS-associated cancer. In one embodiment, the cancer is selected from the group consisting of pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine cancer, thyroid cancer, acute myeloid leukemia (AML), urothelial carcinoma, gastric adenocarcinoma and cervical adenocarcinoma, head and neck squamous cell carcinoma (SCC), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, chronic lymphocytic leukemia (CLL), lung SCC, small cell lung cancer (SCLC), renal papillary carcinoma, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0011] In one aspect, the present application provides a method of inducing an immune response in a subject, the method comprising contacting a cell with a composition comprising a mutant RAS peptide comprising a mutation at a position corresponding to G12 relative to wild-type RAS, thereby stimulating the cell; and administering the stimulated cell to the subject. In one embodiment, the method comprises contacting naive T cells of the subject (T cell, naive T cell) with an antigen presenting cell presenting the mutant RAS peptide, thereby stimulating the T cells. In one embodiment, the cell is autologous to the subject. In one embodiment, the T cells and the antigen presenting cell are autologous to the subject. T cell, naive T cell) with an antigen presenting cell presenting the mutant RAS peptide, thereby stimulating the T cells. In one embodiment, the cell is autologous to the subject. In one embodiment, the T cells and the antigen presenting cell are autologous to the subject.

[0012] In one aspect, the present application provides a composition comprising a T cell receptor (TCR) that specifically binds a mutant RAS (mRAS) peptide in the context of an HLA molecule selected from the group consisting of: HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. In one embodiment, the RAS peptide comprises a mutation at a position corresponding to G12 relative to wild-type RAS. In one embodiment, the mutation of the mRAS peptide corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V relative to wild-type RAS.

[0013] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV39 CDR1, TRAV39 CDR2, TRAV39 CDR3, TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3. In one embodiment, the TCR comprises TRAV39 CDR1, TRAV39 CDR2, TRAV39 CDR3, TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3.

[0014] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV12-1 CDR1, TRAV12-1 CDR2, TRAV12-1 CDR3, TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3. In one embodiment, the TCR comprises TRAV12-1 CDR1, TRAV12-1 CDR2, TRAV12-1 CDR3, TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3.

[0015] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In one embodiment, the TCR comprises TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3.

[0016] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In one embodiment, the TCR comprises TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3.

[0017] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of TRAV19 CDR1, TRAV19 CDR2, TRAV19 CDR3, TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In one embodiment, the TCR comprises TRAV19 CDR1, TRAV19 CDR2, TRAV19 CDR3, TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3.

[0018] In one embodiment, the TCR comprises at least one CDR selected from the group consisting of TRAV4 CDR1, TRAV4 CDR2, TRAV4 CDR3, TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In one embodiment, the TCR comprises TRAV4 CDR1, TRAV4 CDR2, TRAV4 CDR3, TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3.

[0019] In one embodiment, the composition comprises a fusion polypeptide comprising a TCR a chain and a TCR β chain. In one embodiment, the fusion polypeptide comprises a linker domain. In one embodiment, the linker domain is a cleavable linker domain.

[0020] In one aspect, the present application provides a composition comprising an isolated nucleic acid molecule encoding a TCR that specifically binds to a mutant RAS (mRAS) peptide in the context of an HLA molecule selected from the group consisting of HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02.

[0021] In one aspect, the present application provides a cell modified to express a T cell receptor (TCR) that specifically binds to a mutant RAS (mRAS) peptide in the context of an HLA molecule selected from the group consisting of HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. In one embodiment, the mRAS peptide comprises a mutation at a position corresponding to G12 relative to wild-type RAS. In one embodiment, the mutation of the mRAS peptide relative to wild-type RAS corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V.

[0022] In one embodiment, the cell is modified to express a fusion polypeptide comprising a TCR a chain and a TCR β chain. In one embodiment, the cell is genetically modified by introduction of an isolated nucleic acid molecule encoding a polypeptide comprising at least one of a TCR a chain and a TCR β chain. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is selected from the group consisting of a T cell, an NK cell, and an NK T cell. In one embodiment, the cell is autologous to a subject having a RAS-associated cancer. In one embodiment, the cell is autologous to a subject having an HLA type selected from the group consisting of HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02.

[0023] In one aspect, the present application provides a method of treating a subject having a cancer associated with mRAS, the method comprising administering to the subject a cell modified to express a T cell receptor (TCR) that specifically binds to a mutant RAS (mRAS) peptide in the context of an HLA molecule selected from the group consisting of HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, and HLA-B*07:02. In one embodiment, the subject has a cancer selected from the group consisting of pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine cancer, thyroid cancer, acute myeloid leukemia (AML), urothelial carcinoma, gastric adenocarcinoma, and cervical adenocarcinoma, head and neck squamous cell carcinoma (SCC), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, chronic lymphocytic leukemia (CLL), lung SCC, small cell lung cancer (SCLC), renal papillary carcinoma, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0024] In one embodiment, the method comprises identifying the HLA type of the subject. In one embodiment, the method comprises isolating one or more cells of the subject, and modifying the one or more cells to express a TCR. In one embodiment, the method comprises modifying the one or more cells to express a TCR by contacting the one or more cells with an isolated nucleic acid molecule encoding one or more of a TCR a chain and a TCR β chain. BRIEF DESCRIPTION OF DRAWINGS

[0025] The summary above, as well as the detailed description of exemplary embodiments of the application below, will be better understood when read in conjunction with the accompanying drawings. It is understood, however, that the application is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. In the drawings:

[0026] Figure 1 A schematic diagram depicting a discovery strategy for mutant RAS (mRAS) epitopes is depicted.

[0027] Figure 2 A schematic diagram depicting an exemplary computational method for predicting new epitopes of mRAS is depicted.

[0028] Figure 3 Results of an exemplary experiment demonstrating the affinity of predicted mRAS peptides to various HLA molecules are depicted.

[0029] Figure 4A andFigure 4B Results from exemplary experiments demonstrating the predicted affinity of G12 mRAS peptides to various HLA molecules are depicted. Figure 4A A heatmap representing the prediction of mRAS epitopes with <500 nM affinity by computational means using antigen.garnish. Also representing HLA frequencies in the US population and KRAS mutation frequencies occurring in pancreatic adenocarcinoma (PDA), colorectal cancer (CRC) and lung adenocarcinoma (LAC) is depicted. Figure 4B A table summarizing the predicted binding of mRAS epitopes to various HLA molecules is depicted.

[0030] Figure 5 Results from exemplary experiments using a fluorescence polarization assay to determine peptide-MHC binding are depicted. The affinity of various mRAS peptides to specific HLA molecules is shown.

[0031] Figure 6A and Figure 6B Results from exemplary experiments providing a biochemical assessment of mRAS epitope binding are depicted. Figure 6A Competitive peptide binding fluorescence polarization assay. Strong binding affinity is indicated by log[IC50] < 3.7 (dashed line). Figure 6B Peptide stability determined by scintillation proximity assay. Stability of published T cell epitopes is indicated by the grey area (range) and the dashed line (average value).

[0032] Figures 7A-7E Results from experiments using generated monoallelic RAS tandem minigene (TMG) cell lines are depicted. Figure 7A Schematic of lentiviral vector constructs. Figure 7B FACS plot of HLA / RAS TMG modified K56 cell lines indicated by mCherry and GFP positivity. Verification of RAS TMG cell lines by FACS. Figure 7C HLA class I and Figure 7D HLA specific expression. Figure 7E A table of wild type and mRAS long peptide sequences as well as viral control peptides encoded by RAS TMG constructs is depicted.

[0033] Figures 8A-8J Results from exemplary experiments demonstrating the detection of mRAS epitopes by HLA class I immunoprecipitation peptide elution and tandem mass spectrometry (MS / MS) are depicted. Figure 8A A*03:01 restricted KRAS G12D epitope VVV_D. Figure 8B A*03:01 restricted KRAS G12V epitope VV_V. Figure 8C) A*03:01 restricted RAS G12V epitope VVV_V. Figure 8D ) A*03:01 restricted RAS G12R epitope VV_R. Figure 8E ) A*11:01 restricted RAS G12D epitope VV_D. Figure 8F ) A*11:01 restricted RAS G12D epitope VVV_D. Figure 8G ) A*11:01 restricted RAS G12V epitope VVV_V. Figure 8H ) A*11:01 restricted RAS G12V epitope VV_V. Figure 81 ) A*11:01 restricted RAS G12R epitope VV_R. Figure 8J ) B*07:02 restricted RAS G12R epitope GA_R.

[0034] Figure 9 is a schematic depicting a summary of mRAS epitopes detected by mass spectrometry, where shaded boxes represent binding between epitopes and HLA molecules.

[0035] Figure 10 depicts results of exemplary experiments comparing predicted and detected mRAS epitopes in the context of particular HLA types. Peptides highlighted in red are epitopes predicted by computation that were detected using p / MHC IP HPLC tandem mass spectrometry.

[0036] Figure 11 depicts a schematic of experiments detailing a protocol for generating and identifying mRAS-specific CD8+ T cells.

[0037] Figure 12 depicts results of exemplary experiments summarizing mRAS-specific CD8+ T cell responses in healthy donors.

[0038] Figures 13A-13F depicts results of exemplary experiments demonstrating antigenicity of mRAS epitopes. Figure 13A ) A*03:01 restricted, Figure 13B ) A*11:01 restricted, and Figure 13C ) B*07:02 restricted mRAS epitope responses. IFN-γ ELISPOT. Figures 13D-13F ) Representative peptide-MHC multimer staining results for donors are highlighted with red symbols.

[0039] Figure 14 depicts results of exemplary experiments demonstrating detection of mRAS-specific CD8+ by peptide / MHC multimer staining detection.

[0040] Figure 15 Results from exemplary experiments demonstrating that mRAS T cell responses are highly specific for mRAS peptides of interest and do not exhibit any cross-reactivity to wild-type RAS peptides are depicted.

[0041] Figure 16 Results from exemplary experiments demonstrating that B7-G12R responses have high affinity as evidenced by IFN-g secretion and cytotoxicity assays. Importantly, no reactivity was detected against cell lines expressing wild-type or alternatively mutant RAS peptides.

[0042] Figure 17 Results from exemplary experiments demonstrating that HLA-B*07:02 restricted RAS G12R specific CD8+ T cells exhibit cytotoxicity against PSN, a PDA cell line with endogenous RAS G12R expression, when genetically modified to express HLA-B*07:02 are depicted.

[0043] Figure 18 Results from exemplary experiments demonstrating identification of mRAS specific TCR sequences are depicted.

[0044] Figure 19 Design of lentiviral constructs for TCR 831 and TCR 833 are depicted.

[0045] Figure 20 Table summarizing additional TCR constructs, their KRAS mutation specificity, HLA restriction, alpha chain and beta chain identity, and relevant CDR3 amino acid sequences are depicted.

[0046] Figure 21 Results from exemplary experiments demonstrating transgenic expression of TCR 831 and TCR 833 on primary CD8+ cells are depicted.

[0047] Figure 22 Results from exemplary experiments demonstrating that transgenic TCR 831 and TCR 833 have high affinity for HLA-A*11:01 restricted KRAS G12V and no reactivity to wild-type RAS antigens are depicted. Furthermore, TCR 831 and TCR 833 recognize antigen endogenously processed and presented by K562-A*11:01 cells genetically modified to express RASmg constructs.

[0048] Figure 23 Results from exemplary experiments demonstrating that transgenic expression of TCR 831 and TCR 833 confer cytotoxicity to K562-A*11:01 cells expressing endogenous RASmg constructs RAS G12V peptide but not wild-type RAS G12V peptide are depicted.

[0049] Figure 24 Results from an exemplary experiment demonstrating that transgenic expression of TCR831 and TCR833 confers cytotoxicity to Panc03.27, a PDA cell line with endogenous RAS G12V expression, when genetically modified to express HLA-A*11:01.

[0050] Figure 25 A to Figure 25 G depicts results from experiments characterizing TCR831 expression and function. Figure 25 A) TCR expression verified by peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus. Figure 25 B) TCR total avidity assessed by Jurkat reporter cells. Figure 25 C) TCR specificity and cross-reactivity to alternative mutant KRAS epitopes assessed by Jurkat reporter assay. TCR831 exhibits specificity for RAS G12V (VVV_V) but not wild type. Cross-reactivity to RAS G12C (VVV_C) is observed. Figure 25 D) TCR activation of Jurkat reporter cells following co-culture with A*11:01 positive RAS G12V tumor cell lines. Figure 25 E) TCR831 expression on primary CD8+ T cells. Figure 25 F) 4 hour 51Cr assay results demonstrating specific lysis of K562-A*11:01 cells pulsed with G12V peptide (blue) as well as expressing RAS TMG constructs (red) but not wild type (black). Figure 25 G) 4 hour 51Cr assay results demonstrating specific lysis of A*11:01 positive RAS G12V tumor cell lines at an effector to target ratio of 10:1. Cell line colors correspond to Figure 25 C.

[0051] Figure 26 A to Figure 26 G depicts results from exemplary experiments characterizing TCR833 expression and function. Figure 26 A) TCR expression verified by peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus. Figure 26 B) TCR total avidity assessed by Jurkat reporter cells. Figure 26C) Evaluation of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR 831 exhibited specificity to RAS G12V (VVW_V) but not wild type. Cross-reactivity to RAS G12C (VVW_C) was observed. Figure 26 D) TCR activation of Jurkat reporter cells following co-culture with A*11:01 positive RAS G12V tumor cell lines. Figure 26 E) TCR 833 expression on primary CD8+ T cells. Figure 26 F) 4 hour 51Cr assay results demonstrating specific lysis of K562-A*11:01 cells pulsed with G12V peptide (blue) and expressing RAS TMG construct (red) but not wild type (black). Figure 26 G) 4 hour 51Cr assay results demonstrating specific lysis of A*11:01 positive RAS G12V tumor cell lines.

[0052] Figure 27 A to Figure 27 C depicts results of exemplary experiments characterizing TCR 897 expression and function. Figure 27 A) TCR expression verified by peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus. Figure 28 B) TCR total affinity assessed by Jurkat reporter cells. Figure 28 C) Evaluation of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR 897 exhibited specificity to RAS G12V (VVW_V) but not wild type. Cross-reactivity to RAS G12C (VVW_C) and G12D (VVW_D) epitopes was observed.

[0053] Figure 28 A to Figure 28 G depicts results of experiments characterizing TCR 896 expression and function. Figure 28 A) TCR expression verified by peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus. Figure 28 B) TCR total affinity assessed by Jurkat reporter cells. Figure 28 C) Evaluation of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR 896 exhibited specificity to RAS G12V (VVW_V) but not wild type or alternatively mutant KRAS epitopes. Figure 29D) TCR activation of Jurkat reporter cells following co-culture with A*03:01 positive RAS G12V tumor cell lines. Figure 29 E) TCR896 expression on primary CD8+ T cells. Figure 30 F) 4 hour 51Cr assay results demonstrating specific lysis of K562-A*03:01 cells pulsed with G12V peptide (blue) or expressing RAS TMG constructs (red) but not wild type (black). Figure 30 G) 4 hour 51Cr assay results demonstrating specific lysis of A*03:01 positive RAS G12V tumor cell lines.

[0054] Figure 30 A and Figure 30 B depicts results of exemplary experiments characterizing TCR847 expression and function. Figure 30 A) Peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus to verify TCR expression. Example 1: Identification of mRAS neoantigens B) TCR specificity and cross-reactivity to alternative mutant RAS epitopes assessed by Jurkat reporter assay. TCR847 exhibited specificity to RAS G12R (GA_R) but not wild type or alternatively mutant RAS epitopes.

[0055] Figure 1 A to Figure 2 E depicts results of exemplary experiments characterizing TCR864 expression and function. Figure 3 A) Peptide-MHC multimer staining of Jurkat reporter cells transduced by lentivirus to verify TCR expression. Figure 4A B) TCR overall affinity assessed by Jurkat reporter cells. Figure 4B C) TCR specificity and cross-reactivity to alternative mutant RAS epitopes assessed by Jurkat reporter assay. TCR864 exhibited specificity to RAS G12R (GA_R) but not wild type or alternatively mutant RAS epitopes. Figure 4B D) TCR864 expression on primary CD8+ T cells. Figure 5 E) 4 hour 51Cr assay results demonstrating specific lysis of K562-B*07:02 cells pulsed with G12R peptide (blue) or expressing RAS TMG constructs (red) but not wild type (black).

[0056] Figure 5 A schematic of a clinical trial using dendritic cells (DCs) to vaccinate against mRAS short peptides is depicted.

[0057] Figure 6AA schematic depicting the experimental process for identifying mRAS TCRs in vaccinated PDA patients. DETAILED DESCRIPTION

[0058] The present application relates to compositions and methods for treating cancers associated with mutant RAS (mRAS). Somatic mutations within RAS provide a form of non-self antigen, rendering RAS-mutant tumors susceptible to immune-based therapeutic approaches, including but not limited to adoptive T cell therapy. T cells have unique T cell receptors (TCRs) that are capable of recognizing subtle mutations in intracellular proteins that can be expressed and presented on HLA molecules by tumor cells.

[0059] The present application is applicable to any member of the oncogenic protein RAS family, including but not limited to KRAS, NRAS, and HRAS. The RAS hotspot mutations described herein (e.g., mutations at position G12) are common in KRAS, NRAS, and HRAS associated cancers. Furthermore, the amino acid sequences of the RAS peptides described herein are conserved across all RAS family members. Thus, the mutant RAS peptides and TCRs described herein can be applied to induce an immune response against a mutant RAS family member to treat a cancer associated with a mutant RAS family member. As used herein, “RAS” is intended to include any member of the RAS protein family.

[0060] The present application is based in part on the identification of antigenic HLA-restricted mutant RAS peptides. The RAS peptides described herein can be used as immunogenic compositions to induce an immune response against mRAS. In certain embodiments, the present application relates to immunogenic compositions, such as vaccines, comprising an antigenic mRAS peptide described herein or a nucleic acid molecule encoding an antigenic mRAS peptide described herein.

[0061] The present application is based in part on the identification of T cell receptor (TCR) sequences that specifically recognize HLA-restricted mutant RAS antigens. The TCR sequences described herein recognize common mutant RAS antigens in highly prevalent HLA types. In certain aspects, the present application relates to compositions comprising an isolated TCR, or to nucleic acid molecules encoding an isolated TCR, wherein the isolated TCR specifically binds to RAS, mRAS, or a fragment thereof. In one embodiment, the composition comprises a cell, e.g., an autologous or allogeneic T cell, that is genetically modified to express a TCR that specifically binds to RAS, mRAS, or a fragment thereof.

[0062] In certain aspects, the present application relates to methods of treating or preventing an mRAS-associated cancer using an antigenic mRAS peptide or TCR described herein. In one embodiment, the method comprises administering to a subject an immunogenic composition comprising an mRAS peptide or a nucleic acid molecule encoding an mRAS peptide described herein. In one embodiment, the method comprises administering to a subject an immunogenic composition comprising an antigen presenting cell (APC), such as a dendritic cell, that has been loaded with one or more mRAS peptides or one or more nucleic acid molecules encoding one or more mRAS peptides described herein. In certain embodiments, the present application relates to methods of using TCR therapy (e.g., adoptive TCR therapy). In one embodiment, the method comprises administering to a subject having an mRAS-associated cancer at least one T cell that has been genetically modified to express a TCR that specifically binds to RAS, mRAS, or a fragment thereof.

[0063] Exemplary mRAS-associated cancers treatable by the compositions and methods of the present application include, but are not limited to, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine carcinoma, thyroid carcinoma, acute myeloid leukemia (AML), urothelial carcinoma, gastric adenocarcinoma, and cervical adenocarcinoma, head and neck squamous cell carcinoma (SCC), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, chronic lymphocytic leukemia (CLL), lung SCC, small cell lung cancer (SCLC), renal papillary carcinoma, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0064] Figure 6A

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described.

[0066] As used herein, each of the following terms has the meaning associated with it in this section.

[0067] As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0068] The terms "inhibit" and "inhibition" as used herein mean to reduce, suppress, decrease, or block an activity or function by at least about 10% relative to a control value. In some embodiments, the activity is inhibited or blocked by at least about 50% compared to a control value. In some embodiments, the activity is inhibited or blocked by at least about 75%. In some embodiments, the activity is inhibited or blocked by at least about 95%.

[0069] The terms "effective amount" and "pharmaceutically effective amount" refer to an amount of an agent sufficient to provide a desired biological result. That result can be a reduction and / or alleviation of a sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0070] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal (in some embodiments, a mammal, and in some embodiments, a human) having a complement system, including a human in need of or susceptible to therapy for a condition or sequelae thereof. Individuals can include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice, as well as humans.

[0071] The term "abnormal" when used in the context of organisms, tissues, cells, or components thereof, refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells, or components thereof that display the "normal" (expected / homeostatic) respective characteristic. A characteristic that is normal or expected for one cell, tissue type, or subject can be abnormal for a different cell or tissue type.

[0072] "Activated" as used herein refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers to a T cell that, among other things, is undergoing cell division.

[0073] A "disease" is a state of health of a subject in which the subject is unable to maintain homeostasis, and in which the health of the subject continues to deteriorate if the disease is not ameliorated.

[0074] In contrast, a "disorder" of a subject is a state of health in which the subject is able to maintain homeostasis, but the state of health of the subject is less favorable than it would be in the absence of the disorder. A disorder does not necessarily cause the state of health of the subject to further decline if left untreated.

[0075] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which a patient experiences such sign or symptom, or both, are reduced.

[0076] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0077] The term "anti-tumor effect" as used herein refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present application to first prevent the occurrence of a tumor.

[0078] The term "autologous" as used herein is intended to mean any material derived from the same individual to which it will be subsequently re-introduced.

[0079] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0080] "Heterologous" refers to a graft derived from an animal of a different species.

[0081] An "effective amount" or "therapeutically effective amount" of a compound refers to the amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0082] As used herein, "instructional material" includes publications, records, graphs, or any other medium or means that conveys information to the user of the kit concerning the use of the compounds, compositions, vectors, or delivery systems of the present application in the practice of alleviating various diseases or disorders described herein. Alternatively or additionally, the instructional material can describe one or more methods of alleviating a disease or disorder in a cell or tissue of a mammal. For example, the instructional material of the present kit can be attached to the container that holds the identified compounds, compositions, vectors, or delivery systems of the present application, or it can be shipped along with the container holding the identified compounds, compositions, vectors, or delivery systems. Alternatively, the instructional material can be shipped separately from the container, with the intent that the instructional material and the compounds be used cooperatively by the recipient.

[0083] As used herein, "operably linked" or "operatively linked" can mean that the expression of a gene is under the control of a promoter spatially linked thereto. The promoter can be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be approximately equal to the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0084] A "therapeutic treatment" is a treatment administered to a subject who exhibits signs of a disease or disorder, for the purpose of diminishing or eliminating those signs.

[0085] As used herein, "treating a disease or disorder" means reducing the frequency and / or severity of the signs and / or symptoms of the disease or disorder experienced by the patient.

[0086] The phrase "biological sample," "sample," or "specimen" as used herein is intended to include any sample comprising cells, tissues, or bodily fluids in which expression of a nucleic acid or polypeptide can be detected. A biological sample can contain any biological material suitable for detecting a desired biomarker, and can comprise cellular and / or acellular material obtained from an individual. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsies, and smears. Samples that are liquid in nature are referred to herein as "bodily fluids." Biological samples can be obtained from a patient by various techniques, including, for example, by scraping or swabbing an area or by using a needle to obtain a bodily fluid. Methods of collecting various bodily samples are well known in the art.

[0087] "CDR" is defined as the complementarity determining region amino acid sequence of a TCR or TCR chain.

[0088] As used herein, "immunoassay" refers to any binding assay that uses an antibody that is capable of specifically binding to a target molecule to detect and quantify the target molecule.

[0089] The term "specifically binds" as used herein with respect to a polypeptide (e.g., a TCR or TCR chain) means a polypeptide that recognizes and binds a particular target molecule but does not substantially recognize or bind other molecules in a sample. In some instances, the term "specifically binds" or "binds specifically" is used to indicate recognition and binding dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule.

[0090] The "coding region" of a gene consists of the gene coding strand nucleotide residues and the gene non-coding strand nucleotides which are respectively homologous to or complementary to the coding region of an mRNA molecule produced by transcription of the gene.

[0091] The "coding region" of an mRNA molecule also consists of those nucleotide residues of an mRNA molecule that are matched with the anti-codon region of a transfer RNA molecule during translation of the mRNA molecule, or that encode a stop codon. Thus, a coding region can include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0092] "Differentially decreased expression" or "down-regulation" means that the level of a biomarker product is at least 10% or more, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or less, and / or 2.0-fold, 1.8-fold, 1.6-fold, 1.4-fold, 1.2-fold, 1.1-fold or less, and any and all whole or partial increments therebetween, lower than a control.

[0093] "Differentially increased expression" or "up-regulation" means that the level of a biomarker product is at least 10% or more, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, and / or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2.0-fold or more, and any and all whole or partial increments therebetween, higher than a control.

[0094] "Complementary," as used herein with reference to nucleic acids, is a broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairs") with a residue of a second nucleic acid region, and the second nucleic acid region is antiparallel to the first region if the residue is a thymine or a uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand, and the second nucleic acid strand is antiparallel to the first strand if the residue is a guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, a first region comprises a first portion and a second region comprises a second portion, whereby, when the first portion and the second portion are arranged in an antiparallel fashion, at least about 50%, and / or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues of the second portion.

[0095] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0096] "Coding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes. Thus, for example, the specific sequence of nucleotides in a gene or a cDNA encode the sequence of amino acids in a protein by serving as a template for transcription and translation. Both the coding strand, which is used as the template for transcription, and the non-coding strand, which is complementary to the template strand, can be referred to as encoding the protein or other product of the gene or cDNA.

[0097] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate versions of each other, and which encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or an RNA" can also include the introns from which the exons encoding the protein will be spliced away in some forms.

[0098] The term "hybridoma" as used herein refers to a cell produced by the fusion of a B lymphocyte and a fusion partner such as a myeloma cell. The hybridoma can be clonally propagated and maintained in cell culture indefinitely and is capable of producing a monoclonal antibody. A hybridoma can also be considered a hybrid cell.

[0099] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living organism is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated." An isolated nucleic acid or protein can exist in substantially pure form, or can exist in a non-native environment such as, for example, a host cell.

[0100] "Isolated nucleic acid" refers to a nucleic acid segment or fragment which is no longer in the natural state in which it is found in the genome, i.e., a DNA fragment which is no longer contiguous with the sequences which normally flank it in the genome in which it is found. The term also applies to a nucleic acid which has been substantially purified from other components which naturally accompany it in the genome or in a cell, i.e., RNA or DNA or proteins with which it is naturally associated in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0101] In the context of the present invention, the following common nucleic acid base abbreviations are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0102] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. It is general knowledge to the skilled person that a nucleic acid is a polynucleotide which can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means (i.e., cloning of nucleic acid sequences from a recombinant library or the genome of a cell using ordinary cloning techniques and PCR, etc.), as well as by synthetic means.

[0103] "Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are the only viruses in the retrovirus family that can infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, thus they are one of the most effective methods for gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0104] The terms "peptide", "polypeptide", and "protein" are used interchangeably as used herein and refer to a compound made up of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a sequence of a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids linked to one another by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, of which there are numerous types). "Polypeptide" includes, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0105] The term "RNA" as used herein is defined as ribonucleic acid.

[0106] The term "recombinant DNA" as used herein is defined as DNA that is generated by joining DNA fragments from different sources.

[0107] The term "recombinant polypeptide" as used herein is defined as a polypeptide that is generated by using recombinant DNA methods.

[0108] As used herein, “conjugated” refers to the covalent attachment of one molecule to a second molecule.

[0109] “Homologous” refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. Two molecules are homologous at a position in the two sequences if that position in each of the two sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the two molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. The comparison is typically made, for example, by aligning the two sequences and comparing the sequence information, and noting the positions where the sequences match, i.e., where there is a common base or amino acid subunit. Generally, the two sequences are aligned so that maximum homology is obtained.

[0110] The term “variant” as used herein is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or a peptide sequence, respectively, but retains the essential biological properties of the reference molecule. Changes in a nucleic acid variant sequence can not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or can result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in a peptide variant sequence are typically limited or conservative, so that the sequence of the reference peptide and the variant are generally very similar, and identical in many regions. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions, in any combination. A variant of a nucleic acid or a peptide can be naturally occurring, such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be produced by mutagenesis techniques or by direct synthesis. In various embodiments, a variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to a reference sequence.

[0111] The term “modulate” as used herein can mean any method of altering the level or activity of a substrate. Non-limiting examples of modulation with respect to a protein include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting localization of the protein. Non-limiting examples of modulation with respect to an enzyme also include affecting enzymatic activity. A “modulating factor” refers to a molecule whose activity includes affecting the level or activity of a substrate. A modulating factor can be direct or indirect. A modulating factor can act to activate or inhibit or otherwise modulate its substrate.

[0112] As used herein, "scan window" refers to a segment of many consecutive positions in which a sequence can be evaluated independently of any flanking sequence. The scan window is typically incrementally shifted along the length of the sequence to be evaluated, with each new segment being evaluated independently. The incremental shift can be a shift of 1 or more than 1 position.

[0113] As used herein, "vector" can mean a nucleic acid sequence that contains an origin of replication. The vector can be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0114] As used herein, a "substantially pure" cell is a cell that is substantially free of other cell types. A substantially pure cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially pure cells refers to a population of cells that is homogenous. In other instances, this term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0115] RANGES: Throughout this disclosure, various aspects of the application can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the application. Therefore, the description of a range should be considered as an inclusion of each possible sub-range as well as an inclusion of each individual number within that range, for example, within a range of 1 to 6, this would specifically allow for the inclusion of the number 3 and that number 4 unless the context clearly dictates otherwise. This applies regardless of the breadth of the range.

[0116] Figure 6B

[0117] The present application relates to compositions and methods for treating mRAS-related cancers. In various embodiments, the compositions and methods described herein can be used to kill cancer cells, reduce tumor size, inhibit tumor growth, inhibit tumor metastasis, slow tumor progression, or reduce severity, among others.

[0118] In one aspect, the present application relates to immunogenic compositions comprising an antigenic mRAS peptide, wherein the mRAS peptide stimulates or induces an anti-mRAS immune response. In certain embodiments, the mRAS peptide comprises a fragment of mRAS. In certain embodiments, the mRAS peptide comprises an amino acid sequence of about 5-15 amino acids. In certain embodiments, the mRAS peptide comprises an amino acid sequence having a mutation at position G12 relative to wild-type RAS. For example, in one embodiment, the mRAS peptide comprises an amino acid sequence of about 5-15 amino acids and comprises a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS.

[0119] In one aspect, the present application provides an isolated nucleic acid molecule encoding a mRAS peptide described herein. In one aspect, the present application provides a cell, e.g., an antigen presenting cell, comprising a mRAS peptide described herein or a nucleic acid molecule encoding a mRAS peptide.

[0120] In one aspect, the present application relates to compositions comprising a polypeptide comprising one or more TCR chains (e.g., TCR a chain, TCR β chain, TCR δ chain, and TCR γ chain) that specifically bind to RAS, mRAS, or a fragment thereof, alone or together. In one embodiment, the composition comprises a TCR comprising a TCR a chain and a TCR β chain, wherein the TCR specifically binds to RAS, mRAS, or a fragment thereof. Hereinafter, reference to a “TCR” refers to a heterodimeric T cell receptor, a T cell receptor chain alone (e.g., TCR a chain, TCR β chain, TCR δ chain, and TCR γ chain), and functional portions and variants thereof.

[0121] In one embodiment, the TCR specifically binds to mRAS comprising a mutation at position G12 relative to wild-type RAS. For example, in certain embodiments, the TCR specifically binds to mRAS comprising a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS. In certain embodiments, the TCR specifically binds to a fragment of mRAS, wherein the fragment comprises a mutation at a position corresponding to G12. In certain embodiments, the TCR specifically binds to a fragment of mRAS in the context of a particular HLA type. In one embodiment, the composition comprises a fusion polypeptide comprising a TCR a chain and a TCR β chain, wherein the TCR a chain and the TCR β chain together form a heterodimeric TCR. In one embodiment, the fusion polypeptide comprises a cleavable linker between the TCR a chain and the TCR β chain.

[0122] In one aspect, the present application provides an isolated nucleic acid molecule encoding a TCR described herein. In one aspect, the present application provides a cell, such as a T cell, modified to express a TCR described herein.

[0123] In one embodiment, the present application provides a method of treating or preventing an mRAS-associated cancer in a subject having, suspected of having, or at risk of developing an mRAS-associated cancer. Exemplary mRAS-associated cancers that can be treated or prevented by the compositions and methods of the present application include, but are not limited to, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine cancer, thyroid cancer, acute myeloid leukemia (AML), urothelial carcinoma, gastric adenocarcinoma and cervical adenocarcinoma, head and neck squamous cell carcinoma (SCC), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, chronic lymphocytic leukemia (CLL), lung SCC, small cell lung cancer (SCLC), renal papillary carcinoma, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0124] In one embodiment, the method comprises administering to the subject an immunogenic composition comprising an mRAS peptide, a nucleic acid molecule encoding an mRAS peptide, or at least one cell comprising an mRAS peptide or a nucleic acid molecule encoding an mRAS peptide. In one embodiment, the method comprises administering an antigen presenting cell, such as a dendritic cell, loaded with one or more mRAS peptides or one or more nucleic acid molecules encoding one or more mRAS peptides. In some embodiments, the antigen presenting cell is an autologous cell or is derived from an autologous cell. For example, in one embodiment, the method comprises isolating an autologous cell from the subject; culturing the autologous cell ex vivo; loading the isolated autologous cell with one or more mRAS peptides or one or more nucleic acid molecules encoding one or more mRAS peptides, thereby producing an antigen presenting cell presenting an mRAS peptide described herein; and administering the antigen presenting cell to the subject. In certain embodiments, the particular type of mRAS peptide used in the methods of the present application depends on the particular HLA type of the subject or cell.

[0125] In one embodiment, the method comprises administering to the subject a composition comprising a TCR, a nucleic acid molecule encoding a TCR, or at least one cell expressing a TCR, wherein the TCR specifically binds to RAS, mRAS, or a fragment thereof. In one embodiment, the method comprises adoptive TCR therapy, wherein autologous T cells are genetically modified to express a TCR described herein, and administered to the subject to induce an immune response against cancer cells presenting mRAS or a fragment thereof. For example, in one embodiment, the method comprises isolating autologous cells from the subject; culturing the autologous cells ex vivo; genetically modifying the isolated autologous cells to express a TCR described herein; and administering the genetically modified cells to the subject. In certain embodiments, the particular type of TCR used in the methods of the application depends on the particular HLA type of the subject or cells.

[0126] Figure 7A

[0127] In some embodiments, the present application provides compositions comprising an antigenic mRAS peptide. In one embodiment, the mRAS peptide stimulates or induces an anti-mRAS immune response in a subject.

[0128] In one embodiment, the mRAS peptide comprises a mutation at position G12 relative to wild-type RAS. In one embodiment, the mRAS peptide comprises a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS. In certain embodiments, the mRAS peptide is a short fragment of full-length mRAS.

[0129] In one embodiment, the mRAS peptide is about 8 to about 24 amino acid residues, or about 9 to about 11 amino acid residues in length. In one embodiment of the present application, the mRAS peptide comprises a mutation corresponding to G12 relative to wild-type mRAS, and wherein the mRAS peptide is about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 11 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, about 15 amino acid residues, about 16 amino acid residues, about 17 amino acid residues, about 18 amino acid residues, about 19 amino acid residues, about 20 amino acid residues, about 21 amino acid residues, about 22 amino acid residues, about 23 amino acid residues, or about 24 amino acid residues in length.

[0130] Table 1 provides exemplary antigenic mRAS peptides of the present application.

[0131]

[0132] In one embodiment, the present application provides immunogenic compositions for inducing an immune response against mRAS in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. For a composition to be useful as a vaccine, the composition must induce an immune response to mRAS in a cell, tissue, or mammal (e.g., a human). In certain instances, the vaccine induces a protective immune response in a mammal. As used herein, an "immunogenic composition" can comprise an antigen (e.g., an mRAS peptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, or a combination thereof. In particular embodiments, the composition comprises or encodes all or a portion of any of the peptide antigens described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is in admixture with an additional immunostimulatory agent or a nucleic acid encoding such an agent. Immunostimulatory agents include, but are not limited to, additional antigens, immunomodulatory agents, antigen presenting cells, lipid nanoparticles, or adjuvants. In other embodiments, one or more additional agents are covalently bound to the antigen or immunostimulatory agent in any combination.

[0133] In the context of the present application, the term "vaccine" refers to a composition that induces an immune response when inoculated into an animal. In some embodiments, the induced immune response provides protective immunity.

[0134] The vaccines of the present application can differ in their nucleic acid and / or cellular components. In non-limiting examples, vaccines comprising or encoding mRAS peptide antigens can also be formulated with an adjuvant. Of course, it will be appreciated that the various compositions described herein can also comprise additional components. For example, one or more vaccine components can be comprised in a lipid, liposome, or lipid nanoparticle. In another non-limiting example, vaccines can comprise one or more adjuvants. Exemplary adjuvants include, but are not limited to, alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFa, TNFp, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86. Other genes that can be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-I, MadCAM-I, LFA-I, VLA-I, Mac-I, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and functional fragments thereof.

[0135] In accordance with the present disclosure, the vaccines of the present application and their various components can be prepared and / or administered by any of the methods disclosed herein or methods known to one of ordinary skill in the art.

[0136] Induction of immunity by mRAS peptide antigens can be detected by observing the response of all or any portion of the host immune system to mRAS in vivo or in vitro.

[0137] The present application includes cells that have been exposed to an antigen (e.g., an mRAS peptide antigen) or otherwise "pulsed" with an antigen (e.g., an mRAS peptide antigen). For example, antigen presenting cells (APCs), such as dendritic cells (DCs), can be made Ag-loaded in vitro, for example, by culturing ex vivo in the presence of an antigen, or by exposure to an antigen in vivo.

[0138] Those of skill in the art will also readily appreciate that APCs can be "pulsed" in a manner that exposes the APCs to an antigen for a period of time sufficient to promote presentation of the antigen on the surface of the APC. For example, APCs can be exposed to an antigen in the form of small peptide fragments (referred to as antigenic peptides) that are directly "pulsed" onto the exterior of the APC; or the APCs can be incubated with antigenic peptides, which are then taken up by the APCs. The APCs then present the antigenic peptides on the surface of the APCs. Antigens in the form of peptides can be exposed to cells by standard "pulsing" techniques described herein and as known in the art.

[0139] Antigen-loaded APCs (also referred to as "pulsed APCs" of the present application) are generated by exposing APCs to an antigen in vitro or in vivo. In the case of in vitro pulsing of APCs, the APCs can be placed on a culture dish and exposed to an antigen in an amount and for a period of time sufficient to allow the antigen to bind to the APCs. The amount and time required to achieve binding of the antigen to the APCs can be determined by using methods known in the art or disclosed herein. Other methods known to those of skill in the art (e.g., immunoassays or binding assays) can be used to detect the presence of the antigen on the APCs after exposure to the antigen.

[0140] In additional embodiments of the present application, APCs can be transfected with a vector that allows the APCs to express a particular peptide. The peptide expressed by the APCs can then be processed and presented on the cell surface of MHC receptors. The transfected APCs can then be used as an immunogenic composition to generate an immune response against the protein encoded by the vector.

[0141] As discussed elsewhere herein, vectors can be prepared to include a particular polynucleotide that encodes and expresses a peptide against which a desired immunogenic response is desired. In one embodiment, a retroviral vector is used to infect cells. In one embodiment, an adenoviral vector is used to infect cells.

[0142] In another embodiment, the viral vector can be targeted to APCs by modifying the viral vector to encode a protein or portion thereof that is recognized by a receptor on the APC, whereby occupation of the APC receptor by the vector will trigger endocytosis of the vector, allowing processing and presentation of the antigen encoded by the nucleic acid of the viral vector.

[0143] As contemplated herein, various methods can be used to transfect polynucleotides into host cells. The methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, colloidal dispersion systems (i.e., macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes). These methods are understood in the art and are described in the published literature so as to enable one skilled in the art to practice the methods.

[0144] In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector, and the vector can be introduced into an APC to additionally produce a loaded APC. Various types of vectors and methods for introducing nucleic acids into cells are discussed in the available published literature. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). It is readily understood that introduction of an expression vector comprising a polynucleotide encoding an antigen produces a pulsed cell.

[0145] The present application encompasses a variety of methods for pulsing APCs, including, but not limited to, loading APCs with peptide antigens or with cDNA or mRNA encoding peptide antigens. However, the present application should not be construed as being limited to particular forms of antigens for pulsing APCs. Rather, the present application encompasses other methods known in the art for producing antigen-loaded APCs. In one embodiment, APCs are transfected with mRNA encoding a defined antigen. Using appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with a transcription reaction, mRNA corresponding to a gene product whose sequence is known can be rapidly produced in vitro. Transfecting APCs with mRNA offers advantages over other antigen loading techniques for producing pulsed APCs. For example, the ability to amplify RNA from minute amounts of tissue (i.e., tumor tissue) extends the use of APCs for vaccination to a large number of patients.

[0146] There are a number of methods that can be used to engineer DCs and other APCs, such as mRNA-based delivery, DNA plasmid-based delivery, all of which are encompassed by the present application. That is, any delivery system can be used to engineer immune cells to express the mRAS peptides described herein.

[0147] It will be appreciated that the antigenic compositions of the present application can be prepared by methods well known in the art, including but not limited to chemical synthesis by solid phase synthesis and purification from other products of the chemical reaction by HPLC, or by expressing a nucleic acid sequence (e.g., a DNA sequence) encoding a peptide antigen of the present application in an in vitro translation system or in a living cell. In addition, the antigenic compositions can comprise cellular components isolated from a biological sample. The antigenic compositions are isolated and extensively dialyzed to remove one or more small molecular weight molecules that are not desired and / or lyophilized so as to more easily be formulated into a desired carrier. It will also be appreciated that additional amino acids, mutations, chemical modifications, etc., if any, made in preparing the vaccine components do not substantially interfere with the antibody recognition of the epitope sequences. The peptide sequences can be synthesized by methods known to those of ordinary skill in the art, for example, peptide synthesis using an automated peptide synthesizer, such as those available from Applied Biosystems, Inc., Foster City, CA.

[0148] Longer peptides or polypeptides can also be prepared, for example, by recombinant means. In certain embodiments, a nucleic acid encoding an antigenic composition and / or component described herein can be used, for example, to produce an antigenic composition in vitro or in vivo for use in the various compositions and methods of the present application. For example, in certain embodiments, a nucleic acid encoding an antigen is contained in a vector, for example, in a recombinant cell. The nucleic acid can be expressed to produce a peptide or polypeptide comprising the antigenic sequence. The peptide or polypeptide can be secreted from the cell, or contained as part of the cell or within the cell.

[0149] In certain embodiments, an immune response can be facilitated by transfecting or inoculating a mammal with a nucleic acid encoding an antigen. Following administration of the nucleic acid to the mammal, one or more cells contained in the target mammal express the sequence encoded by the nucleic acid. The vaccine can also be in the form of a nucleic acid (e.g., cDNA or RNA) encoding all or a portion of a peptide or polypeptide sequence of an antigen, for example. In vivo expression of the nucleic acid can be, for example, by a plasmid-type vector, a viral vector, or a viral / plasmid construct vector.

[0150] In another embodiment, the nucleic acid comprises a coding region comprising all or a portion of a sequence encoding an appropriate antigen or immunologically functional equivalent thereof. Of course, the nucleic acid can comprise and / or encode additional sequences, including but not limited to sequences comprising one or more immunomodulators or adjuvants.

[0151] In certain embodiments, the immunological composition comprises immune cells stimulated by APCs loaded with or pulsed with one or more mRAS peptide antigens described herein. For example, in one embodiment, the immunological composition comprises stimulated T cells that were cultured with and activated by APCs loaded with or pulsed with one or more mRAS peptide antigens described herein. In one embodiment, the stimulated cells are derived from naive cells (e.g., naive T cells) that were then cultured with and activated by APCs loaded with or pulsed with one or more mRAS peptide antigens described herein. In certain embodiments, the naive cells are autologous or allogeneic to the ultimate recipient of the stimulated cells. In one embodiment, both the naive cells and the APCs are from the same subject. In one embodiment, both the naive cells and the APCs are from different subjects within the same species.

[0152] Methods for detecting cytotoxic T lymphocyte induction are well known. Foreign substances entering the living body are presented to T cells and B cells by the action of APCs. In response to the stimulation of antigens, T cells that react to antigens presented by APCs in an antigen-specific manner differentiate into cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs). These antigen-stimulated cells then proliferate. This process is referred to herein as the "activation" of T cells. Thus, CTL induction by an epitope of a polypeptide or peptide or combination thereof can be assessed by presenting the epitope of the polypeptide or peptide or combination thereof to T cells by APCs and detecting the induction of CTLs. In addition, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0153] Methods for using dendritic cells (DCs) as APCs to assess the induction of CTLs are well known in the art. DCs are representative APCs that have a robust CTL-inducing effect among APCs. In the methods of the present application, an epitope of a polypeptide or peptide or combination thereof is initially expressed by a DC, and then the DC is contacted with T cells. Detecting T cells that have a cytotoxic effect on cells of interest after contact with the DC indicates that the epitope of the polypeptide or peptide or combination thereof has the activity of inducing cytotoxic T cells. In addition, the induced immune response can also be examined by measuring IFN-γ produced and released by CTLs in the presence of antigen-presenting cells carrying immobilized peptides or peptide combinations by visualization using anti-IFN-γ antibodies, such as ELISPOT assays.

[0154] In addition to DCs, peripheral blood mononuclear cells (PBMCs) can also be used as APCs. It has been reported that the induction of CTLs is enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTLs have been shown to be induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0155] Antigens that are confirmed to have CTL-inducing activity by these methods are antigens that have a DC-activating effect and subsequent CTL-inducing activity. Furthermore, CTLs that acquire cytotoxicity through antigen presentation by APCs can also be used as vaccines against antigen-related disorders.

[0156] The induction of immunity by expression of an mRAS peptide antigen can be further confirmed by observing the induction of antibody production against mRAS. For example, a composition is determined to induce immunity when antibodies against an antigen are induced in a laboratory animal immunized with the composition encoding the antigen, and when an antigen-related pathology is inhibited by those antibodies.

[0157] The induction of immunity by expression of an mRAS peptide antigen can be further confirmed by observing the induction of CD4+ T cells. CD4+ T cells can also lyse target cells, but primarily provide help in inducing other types of immune responses, including CTLs and antibody production. The type of CD4+ T cell help can be characterized as Thl, Th2, Th9, Thl7, regulatory T, or T follicular helper (Tfh) cells. Each subtype of CD4+ T cell provides help for certain types of immune responses. In one embodiment, the composition selectively induces T follicular helper cells, which drive potent antibody responses. fh ) cells. Each subtype of CD4+ T cell provides help for certain types of immune responses. In one embodiment, the composition selectively induces T follicular helper cells, which drive potent antibody responses.

[0158] Figure 7E

[0159] In some embodiments, the present application provides compositions comprising a polypeptide that specifically binds to RAS, mRAS, or a fragment thereof. In one embodiment, the polypeptide comprises a TCR that specifically binds to RAS, mRAS, or a fragment thereof in the context of a particular HLA type.

[0160] In one embodiment, the TCR specifically binds to mRAS comprising a mutation at position G12 relative to wild-type RAS. For example, in certain embodiments, the TCR specifically binds to mRAS comprising a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS. In certain embodiments, the TCR specifically binds to a fragment of mRAS, wherein the fragment comprises a mutation at a position corresponding to G12.

[0161] In one embodiment of the application, as described above, the TCR has antigenic specificity for an mRAS peptide having a mutation at G12, the mRAS peptide having any length. For example, the TCR can have antigenic specificity for an mRAS peptide having a mutation corresponding to G12, the mRAS peptide having a length of about 8 to about 24 amino acid residues, or about 9 to about 11 amino acid residues. In one embodiment of the application, the TCR can have antigenic specificity for an mRAS peptide having a mutation corresponding to G12, the mRAS peptide having a length of about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 11 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, about 15 amino acid residues, about 16 amino acid residues, about 17 amino acid residues, about 18 amino acid residues, about 19 amino acid residues, about 20 amino acid residues, about 21 amino acid residues, about 22 amino acid residues, about 23 amino acid residues, or about 24 amino acid residues. Exemplary mRAS peptides having a mutation corresponding to G12 to which a TCR specifically binds can be found in Table 1.

[0162] In certain embodiments, the TCR specifically binds to an mRAS peptide in the context of a particular HLA molecule. The HLA molecule corresponding to the mRAS peptide can be found in Table 1.

[0163] Figure 7C

[0164] In one embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at a position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA C GV (SEQ ID NO: 1) in the context of an HLA-A*02:01 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA C GV (SEQ ID NO: 1) in the context of an HLA-A*02:01 molecule.

[0165] Figure 7D

[0166] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at the position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA D GV (SEQ ID NO: 2) in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA D GV (SEQ ID NO: 2).

[0167] Figures 8A-8J

[0168] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA R GV (SEQ ID NO: 3) in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA R GV (SEQ ID NO: 3).

[0169] Figure 9

[0170] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA V GV (SEQ ID NO: 4) in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising KLVVVGA V GV (SEQ ID NO: 4).

[0171] Figure 10

[0172] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at the position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA C GVGK (SEQ ID NO: 5) or VVVGA C GVGK (SEQ ID NO: 6) in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA C GVGK (SEQ ID NO: 5) or VVVGA C GVGK (SEQ ID NO: 6) in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA

[0173] Example 2: Assessment of immunogenicity of mRAS

[0174] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at the position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8) in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8) in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA

[0175] Figure 11

[0176] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA R GVGK (SEQ ID NO: 9) or VVVGA RmRAS peptide of GVGK (SEQ ID NO: 10). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA R mRAS peptide of GVGK (SEQ ID NO: 9) or VVGA R mRAS peptide of GVGK (SEQ ID NO: 10).

[0177] Figure 12

[0178] In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA V mRAS peptide of GVGK (SEQ ID NO: 11) or VVGA V mRAS peptide of GVGK (SEQ ID NO: 12). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA V mRAS peptide of GVGK (SEQ ID NO: 11) or VVGA V mRAS peptide of GVGK (SEQ ID NO: 12).

[0179] Figures 13A-13F

[0180] In one embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at the position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12C mutation at the position corresponding to RAS G12 in the context of an HLA-A*03:01 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA C mRAS peptide of GVGK (SEQ ID NO: 5) or VVGA C mRAS peptide of GVGK (SEQ ID NO: 6). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA C mRAS peptide of GVGK (SEQ ID NO: 5) or VVGA C mRAS peptide of GVGK (SEQ ID NO: 6).

[0181] Figure 14

[0182] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at the position corresponding to RAS G12, in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8), in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8), in the context of an HLA-A*02:01 molecule.

[0183] Figure 15

[0184] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12, in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA R GVGK (SEQ ID NO: 9) or VVVGA R GVGK (SEQ ID NO: 10), in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA R GVGK (SEQ ID NO: 9) or VVVGA R GVGK (SEQ ID NO: 10), in the context of an HLA-A*02:01 molecule.

[0185] Figure 16

[0186] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12, in the context of an HLA-A*02:01 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) or VVVGA VmRAS peptide of GVGK (SEQ ID NO: 12). For example, in one embodiment, the TCR specifically binds an mRAS peptide comprising VVGA V mRAS peptide of GVGK (SEQ ID NO: 11) or VVGA V mRAS peptide of GVGK (SEQ ID NO: 12).

[0187] Figure 17

[0188] In one embodiment, the TCR specifically binds an mRAS peptide having a G12C mutation at the position corresponding to RAS G12. In one embodiment, the TCR specifically binds an mRAS peptide having a G12C mutation at the position corresponding to RAS G12 in the context of an HLA-B*07:02 molecule. For example, in one embodiment, the TCR specifically binds an mRAS peptide comprising GA C mRAS peptide of GVGKSAL (SEQ ID NO: 13). For example, in one embodiment, the TCR specifically binds an mRAS peptide comprising GA C mRAS peptide of GVGKSAL (SEQ ID NO: 13).

[0189] Example 3: Development of mRAS-specific TCR therapy

[0190] In one embodiment, the TCR specifically binds an mRAS peptide having a G12D mutation at the position corresponding to RAS G12. In one embodiment, the TCR specifically binds an mRAS peptide having a G12D mutation at the position corresponding to RAS G12 in the context of an HLA-B*07:02 molecule. For example, in one embodiment, the TCR specifically binds an mRAS peptide comprising GA D mRAS peptide of GVGKSAL (SEQ ID NO: 14). For example, in one embodiment, the TCR specifically binds an mRAS peptide comprising GA D mRAS peptide of GVGKSAL (SEQ ID NO: 14).

[0191] Figure 18

[0192] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 in the context of an HLA-B*07:02 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising GA R GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising GA R GVGKSAL (SEQ ID NO: 15).

[0193] Figure 19

[0194] In an embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12. In an embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at the position corresponding to RAS G12 in the context of an HLA-B*07:02 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising GA V GVGKSAL (SEQ ID NO: 16) in the context of an HLA-B*07:02 molecule. For example, in an embodiment, the TCR specifically binds to an mRAS peptide comprising GA V GVGKSAL (SEQ ID NO: 16).

[0195] Figure 20

[0196] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at the position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0197] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at the position corresponding to RAS G12 comprises a T cell receptor alpha variable region 39 (TRAV39-01*01; also referred to herein as “TRAV39”) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at the position corresponding to RAS G12 comprises a TRAV39 CDR1, wherein the TRAV39 CDR1 comprises the following amino acid sequence: STTSDRL (SEQ ID NO: 17).

[0198] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV39 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV39 CDR2, wherein the TRAV39 CDR2 comprises the following amino acid sequence: VLLSNGAVK (SEQ ID NO: 18).

[0199] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV39 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV39 CDR3, wherein the TRAV39 CDR3 comprises the following amino acid sequence: CAVDKDGGYQKVTF (SEQ ID NO: 19).

[0200] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3.

[0201] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV39. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV39, wherein the variable domain of TRAV39 comprises the following amino acid sequence: ELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAVHDLSATYFCAVDKDGGYQKVTFGTGTKLQVIP (SEQ ID NO: 20).

[0202] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence:

[0203] In one embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence:

[0204] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0205] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable region 20-1 (TRBV20-1*01; also referred to herein as “TRBV20”) CDR1. In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR1, wherein the TRBV20-1 CDR1 comprises the following amino acid sequence: LDFQATTM (SEQ ID NO: 23).

[0206] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR2. In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR2, wherein the TRBV20-1 CDR2 comprises the following amino acid sequence: TSNEGSKAT (SEQ ID NO: 24).

[0207] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR3. In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR3, wherein the TRBV20-1 CDR3 comprises the following amino acid sequence: CSASPRAGQLSSYNSPLHF (SEQ ID NO: 25).

[0208] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR1, a TRBV20-1 CDR2, and a TRBV20-1 CDR3.

[0209] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV20-1. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV20-1, wherein the variable domain of TRBV20-1 comprises the following amino acid sequence: GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASPRAGQLSSYNSPLHFGNGTRLTV (SEQ ID NO: 26).

[0210] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence:

[0211] In one embodiment, the TCR comprises a TCR beta chain comprising the following amino acid sequence:

[0212] In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR β chain comprising one or more of TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR β chain comprising one or more of TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3, and binds to an mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR β chain comprising one or more of TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3, and binds to an mRAS peptide comprising VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR β chain comprising one or more of TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3, and binds to an mRAS peptide comprising VVVGA C GVGK (SEQ ID NO: 6) in the context of an HLA-A*11:01 molecule.

[0213] In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3, and (b) a TCR β chain comprising a TRBV20-1 CDR1, a TRBV20-1 CDR2, and a TRBV20-1 CDR3. In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3, and (b) a TCR β chain comprising a TRBV20-1 CDR1, a TRBV20-1 CDR2, and a TRBV20-1 CDR3, and binds to an mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3, and (b) a TCR β chain comprising a TRBV20-1 CDR1, a TRBV20-1 CDR2, and a TRBV20-1 CDR3, and binds to an mRAS peptide comprising VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3, and (b) a TCR β chain comprising a TRBV20-1 CDR1, a TRBV20-1 CDR2, and a TRBV20-1 CDR3, and binds to an mRAS peptide comprising VVVGA C GVGK (SEQ ID NO: 6) in the context of an HLA-A*11:01 molecule.

[0214] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds to an mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds to an mRAS peptide comprising VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds to an mRAS peptide comprising VVVGA Ca mRAS peptide of GVGK (SEQ ID NO: 6).

[0215] In one embodiment, a TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises all of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds to a mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds to a mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12 in the context of an HLA-A*11:01 molecule. Va mRAS peptide of GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds a peptide comprising VVVGA C a mRAS peptide of GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV39-CDR1: SEQ ID NO: 17; TRAV39-CDR2: SEQ ID NO: 18; TRAV39-CDR3: SEQ ID NO: 19; TRBV20-1-CDR1: SEQ ID NO: 23; TRBV20-1-CDR2: SEQ ID NO: 24; and TRBV20-1-CDR3: SEQ ID NO: 25, or one or more variants thereof, and binds a peptide comprising VVVGA

[0216] In one embodiment, the composition comprises a fusion protein comprising a TCR a chain and a TCR β chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR a chain from the TCR β chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 29).

[0217] In one embodiment, the composition comprises a fusion protein comprising an amino acid sequence of:

[0218]

[0219] Figure 20

[0220] In one embodiment, a TCR that specifically binds a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR a chain.

[0221] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor alpha variable region 12-1 (TRAV12-1*01; also referred to herein as “TRAV12-1”) CDR1. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR1, wherein the TRAV12-1 CDR1 comprises the following amino acid sequence: SNSASQSF (SEQ ID NO: 31).

[0222] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR2, wherein the TRAV12-1 CDR2 comprises the following amino acid sequence: SVYSSGNE (SEQ ID NO: 32).

[0223] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR3, wherein the TRAV12-1 CDR3 comprises the following amino acid sequence: CAVNPPDTGFQKLVF (SEQ ID NO: 33).

[0224] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV12-1 CDR1, a TRAV12-1 CDR2, and a TRAV12-1 CDR3.

[0225] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV12-1. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV12-1, wherein the variable domain of TRAV12-1 comprises the following amino acid sequence: RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVNPPDTGFQKLVFGTGTRLLVSP (SEQ ID NO: 34).

[0226] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 35).

[0227] In one embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence: MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVNPPDTGFQKLVFGTGTRLLVSPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 36).

[0228] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0229] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable 28 (TRBV28*01; also referred to herein as “TRBV28”) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR1, wherein the TRBV28 CDR1 comprises the following amino acid sequence: DMDHENM (SEQ ID NO: 37).

[0230] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR2, wherein the TRBV28 CDR2 comprises the following amino acid sequence: FSYDVKME (SEQ ID NO: 38).

[0231] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR3, wherein the TRBV28 CDR3 comprises the following amino acid sequence: CASSLSFRQGLREQYF (SEQ ID NO: 39).

[0232] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3.

[0233] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV28. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV28, wherein the variable domain of TRBV28 comprises the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSFRQGLREQYFGPGTRLTVT (SEQ ID NO: 40).

[0234] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 41).

[0235] In an embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence of: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSFRQGLREQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 42)

[0236] In an embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of a TRAV12-1 CDR1, a TRAV12-1 CDR2, and a TRAV12-1 CDR3, and (b) a TCR beta chain comprising one or more of a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3. In an embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of a TRAV12-1 CDR1, a TRAV12-1 CDR2, and a TRAV12-1 CDR3, and (b) a TCR beta chain comprising one or more of a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3, and binds to a mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of a TRAV12-1 CDR1, a TRAV12-1 CDR2, and a TRAV12-1 CDR3, and (b) a TCR beta chain comprising one or more of a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3, and binds to a peptide comprising VVVGA Va mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising VVVGA C a mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising VVVGA

[0237] In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising VVVGA V a mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising VVVGA C a mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds a mRAS peptide comprising VVVGA

[0238] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds to an mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds to an mRAS peptide comprising VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds to an mRAS peptide comprising VVVGA Ca mRAS peptide of GVGK (SEQ ID NO: 6).

[0239] In one embodiment, a TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises all of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds to a mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV12-1-CDR1: SEQ ID NO: 31; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1: SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds to a mRAS peptide comprising a G12V or G12C mutation at a position relative to RAS G12 in the context of an HLA-A*11:01 molecule. Va mRAS peptide of GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV12-1-CDR1 : SEQ ID NO: 31 ; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1 : SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds, in the context of an HLA-A*11 :01 molecule, a mRAS peptide comprising VVVGA C a mRAS peptide of GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV12-1-CDR1 : SEQ ID NO: 31 ; TRAV12-1-CDR2: SEQ ID NO: 32; TRAV12-1-CDR3: SEQ ID NO: 33; TRBV28-CDR1 : SEQ ID NO: 37; TRBV28-CDR2: SEQ ID NO: 38; and TRBV28-CDR3: SEQ ID NO: 39, or one or more variants thereof, and binds, in the context of an HLA-A*11 :01 molecule, a mRAS peptide comprising VVVGA

[0240] In one embodiment, the composition comprises a fusion protein comprising a TCR a chain and a TCR β chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR a chain from the TCR β chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 43).

[0241] In one embodiment, the composition comprises a fusion protein comprising an amino acid sequence of: MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVNPPDTGFQKLVFGTGTRLLVSPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGEGRGSLLTCGDVEENPGPMGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSFRQGLREQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 44).

[0242] Figure 21

[0243] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0244] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor alpha variable region 17 (TRAV17) CDR1. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR1, wherein the TRAV17 CDR1 comprises the following amino acid sequence: KTSINNL (SEQ ID NO: 45).

[0245] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR2, wherein the TRAV17 CDR2 comprises the following amino acid sequence: LIRSNEREK (SEQ ID NO: 46).

[0246] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR3, wherein the TRAV17 CDR3 comprises the following amino acid sequence: CATDPGGFKTIF (SEQ ID NO: 47).

[0247] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3.

[0248] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV17. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV17, wherein the variable domain of TRAV17 comprises the following amino acid sequence: SQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATD (SEQ ID NO: 169).

[0249] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 48).

[0250] In an embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 49).

[0251] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0252] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable region 11-2 (TRBV11-2) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR1, wherein the TRBV11-2 CDR1 comprises the following amino acid sequence: ISGHATL (SEQ ID NO: 50).

[0253] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR2, wherein the TRBV11-2 CDR2 comprises the following amino acid sequence: QFQNNGVV (SEQ ID NO: 51).

[0254] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR3, wherein the TRBV11-2 CDR3 comprises the following amino acid sequence: CASSLYGGSISYEQYF (SEQ ID NO: 52).

[0255] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR1, a TRBV11-2 CDR2, and a TRBV11-2 CDR3.

[0256] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV11-2 CDR2, wherein the TRBV11-2 CDR2 comprises the following amino acid sequence: QFQNNGVV (SEQ ID NO: 51).

[0257] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 53).

[0258] In an embodiment, the TCR comprises a TCR beta chain comprising the following amino acid sequence: MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLYGGSISYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 54).

[0259] In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds to an mRAS peptide comprising a G12V, G12C, or G12D mutation at a position relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds to an mRAS peptide comprising VVGA C GVGK (SEQ ID NO: 5) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds to an mRAS peptide comprising VVGA Da mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7) in the context of an HLA-A*11:01 molecule. R a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7) in the context of an HLA-A*11:01 molecule.

[0260] In one embodiment, the TCR comprises (a) a TCR a chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In one embodiment, the TCR comprises (a) a TCR a chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds a mRAS peptide comprising a G12V, G12C, or G12D mutation at a position relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR a chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7) in the context of an HLA-A*11:01 molecule. V a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds a mRAS peptide comprising VVGA GVGK (SEQ ID NO: 7) in the context of an HLA-A*11:01 molecule. Ca mRAS peptide of GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3, and (b) a TCR β chain comprising a TRBV11-2 CDR1, a TRBV11-2 CDR2, and a TRBV11-2 CDR3, and binds, in the context of an HLA-A*11:01 molecule, a peptide comprising VVGA D a mRAS peptide of GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises (a) a TCR a chain comprising a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3, and (b) a TCR β chain comprising a TRBV11-2 CDR1, a TRBV11-2 CDR2, and a TRBV11-2 CDR3, and binds, in the context of an HLA-A*11:01 molecule, a peptide comprising VVGA R a mRAS peptide of GVGK (SEQ ID NO: 9).

[0261] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising a G12V, G12C, or G12D mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) in the context of an HLA-A* 11 :01 molecule. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising VVGA Ca mRAS peptide of GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1 : SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 46; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1 : SEQ ID NO: 50; TRBV11-2-CDR2: SEQ ID NO: 51 ; and TRBV11-2-CDR3: SEQ ID NO: 52, or one or more variants thereof, and binds, in the context of an HLA-A*11 :01 molecule, to a peptide comprising VVGA D a mRAS peptide of GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1 : SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 46; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1 : SEQ ID NO: 50; TRBV11-2-CDR2: SEQ ID NO: 51 ; and TRBV11-2-CDR3: SEQ ID NO: 52, or one or more variants thereof, and binds, in the context of an HLA-A*11 :01 molecule, to a peptide comprising VVGA R a mRAS peptide of GVGK (SEQ ID NO: 9).

[0262] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising a G12V, G12C, or G12D mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) in the context of an HLA-A*11:01 molecule. In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:46; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:50; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or one or more variants thereof, and binds to an mRAS peptide comprising VVGA Ca mRAS peptide of GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 46; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1: SEQ ID NO: 50; TRBV11-2-CDR2: SEQ ID NO: 51; and TRBV11-2-CDR3: SEQ ID NO: 52, or one or more variants thereof, and binds a peptide comprising VVGA D a mRAS peptide of GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 46; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1: SEQ ID NO: 50; TRBV11-2-CDR2: SEQ ID NO: 51; and TRBV11-2-CDR3: SEQ ID NO: 52, or one or more variants thereof, and binds a peptide comprising VVGA R a mRAS peptide of GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises all of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 46; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1: SEQ ID NO: 50; TRBV11-2-CDR2: SEQ ID NO: 51; and TRBV11-2-CDR3: SEQ ID NO: 52, or one or more variants thereof, and binds a peptide comprising VVGA

[0263] In one embodiment, the composition comprises a fusion protein comprising a TCR a chain and a TCR β chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR a chain from the TCR β chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 55).

[0264] In one embodiment, the composition comprises a fusion protein comprising an amino acid sequence of: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGEGRGSLLTCGDVEENPGPMGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLYGGSISYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 56).

[0265] Figure 22

[0266] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12V mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0267] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor alpha variable region 19 (TRAV19) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR1, wherein the TRAV19 CDR1 comprises the following amino acid sequence: ETRDTTYYL (SEQ ID NO: 57).

[0268] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR2, wherein the TRAV19 CDR2 comprises the following amino acid sequence: RRNSFDEQNE (SEQ ID NO: 58).

[0269] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR3, wherein the TRAV19 CDR3 comprises the following amino acid sequence: CALSEAGTYKYIF (SEQ ID NO: 59).

[0270] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR1, a TRAV19 CDR2, and a TRAV19 CDR3.

[0271] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV19. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV19, wherein the variable domain of TRAV19 comprises the following amino acid sequence: AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSE (SEQ ID NO: 171).

[0272] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 60).

[0273] In an embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence: MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 61).

[0274] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0275] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable region 9 (TRBV9) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR1, wherein the TRBV9 CDR1 comprises the following amino acid sequence: RSGDLSV (SEQ ID NO: 62).

[0276] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR2, wherein the TRBV9 CDR2 comprises the following amino acid sequence: QYYNGEER (SEQ ID NO: 63).

[0277] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR3, wherein the TRBV9 CDR3 comprises the following amino acid sequence: CASSVAGGGQETQYF (SEQ ID NO: 64).

[0278] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR1, a TRBV9 CDR2, and a TRBV9 CDR3.

[0279] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV9. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV9, wherein the variable domain of TRBV9 comprises the following amino acid sequence: DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSV (SEQ ID NO: 172).

[0280] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 65).

[0281] In an embodiment, the TCR comprises a TCR beta chain comprising the following amino acid sequence: MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVAGGGQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 66).

[0282] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising a G12V mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) or VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*03:01 molecule.

[0283] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising a G12V mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising VVGA V GVGK (SEQ ID NO: 11) or VVVGA V GVGK (SEQ ID NO: 12) in the context of an HLA-A*03:01 molecule.

[0284] In one embodiment, the TCR that specifically binds to an mRAS peptide with a G12 mutation at a position corresponding to RAS G12 comprises at least one CDR selected from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variations thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof, and is combined with an mRAS peptide containing a G12V mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof, and is bound to a VVGA-containing compound in the context of an HLA-A*03:01 molecule. V GVGK (SEQ ID NO:11) or VVVGA V mRAS peptide of GVGK (SEQ ID NO:12).

[0285] In another embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises all of the CDRs from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof, and binds to an mRAS peptide comprising a G12V mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof, and binds to an mRAS peptide comprising a G12C mutation at a position relative to RAS G12 in the context of an HLA-A*02:01 molecule. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV19-CDR1: SEQ ID NO: 57; TRAV19-CDR2: SEQ ID NO: 58; TRAV19-CDR3: SEQ ID NO: 59; TRBV9-CDR1: SEQ ID NO: 62; TRBV9-CDR2: SEQ ID NO: 63; and TRBV9-CDR3: SEQ ID NO: 64, or one or more variants thereof, and binds to an mRAS peptide comprising a G12S mutation at a position relative to RAS G12 in the context of an HLA-A*02:01 molecule. V GVGK (SEQ ID NO: 11) or VVGA V GVGK (SEQ ID NO: 12).

[0286] In one embodiment, the composition comprises a fusion protein comprising a TCR alpha chain and a TCR beta chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha chain from the TCR beta chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 67).

[0287] In one embodiment, the composition comprises a fusion protein comprising an amino acid sequence of: MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGEGRGSLLTCGDVEENPGPMGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVAGGGQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 68).

[0288] Figure 22

[0289] In one embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0290] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor alpha variable region 17 (TRAV17) CDR1. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR1, wherein the TRAV17 CDR1 comprises the following amino acid sequence: KTSINNL (SEQ ID NO: 69).

[0291] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR2, wherein the TRAV17 CDR2 comprises the following amino acid sequence: LIRSNEREK (SEQ ID NO: 70).

[0292] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR3, wherein the TRAV17 CDR3 comprises the following amino acid sequence: CATFPNFGNEKLTF (SEQ ID NO: 71).

[0293] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3.

[0294] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV17. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV17, wherein the variable domain of TRAV17 comprises the following amino acid sequence: SQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATF (SEQ ID NO: 173).

[0295] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 72).

[0296] In an embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATFPNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 73).

[0297] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0298] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable region 10-3 (TRBV10-3) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR1, wherein the TRBV10-3 CDR1 comprises the following amino acid sequence: TENHRYM (SEQ ID NO: 74).

[0299] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR2, wherein the TRBV10-3 CDR2 comprises the following amino acid sequence: YSYGVKDT (SEQ ID NO: 75).

[0300] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR3, wherein the TRBV10-3 CDR3 comprises the following amino acid sequence: CAISESERYYEQYF (SEQ ID NO: 76).

[0301] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR1, a TRBV10-3 CDR2, and a TRBV10-3 CDR3.

[0302] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV10-3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV10-3, wherein the variable domain of TRBV10-3 comprises the following amino acid sequence: DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISE (SEQ ID NO: 174).

[0303] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 77).

[0304] In an embodiment, the TCR comprises a TCR beta chain comprising the following amino acid sequence: MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISESERYYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 78).

[0305] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising G12R at the position relative to RAS G12 in the context of an HLA-B*07:02 molecule. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule. R GVGKSAL (SEQ ID NO: 15).

[0306] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising G12R at the position relative to RAS G12 in the context of an HLA-B*07:02 molecule. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule. R GVGKSAL (SEQ ID NO: 15).

[0307] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule, wherein the mRAS peptide comprises the sequence of GVGKSAL (SEQ ID NO: 15). R GVGKSAL (SEQ ID NO: 15).

[0308] In another embodiment, the TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises all of the CDRs from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV17-CDR1: SEQ ID NO: 69; TRAV17-CDR2: SEQ ID NO: 70; TRAV17-CDR3: SEQ ID NO: 71; TRBV10-3-CDR1: SEQ ID NO: 74; TRBV10-3-CDR2: SEQ ID NO: 75; and TRBV10-3-CDR3: SEQ ID NO: 76, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule, wherein the mRAS peptide has the amino acid sequence of: GVGKSAL(SEQ ID NO: 15). R GVGKSAL (SEQ ID NO: 15).

[0309] In one embodiment, the composition comprises a fusion protein comprising a TCR alpha chain and a TCR beta chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha chain from the TCR beta chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence of: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 79).

[0310] In an embodiment, the composition comprises a fusion protein comprising an amino acid sequence of: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATFPNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGEGRGSLLTCGDVEENPGPMGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISESERYYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 80).

[0311] Figure 23

[0312] In an embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0313] In an embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor alpha variable region 4 (TRAV4) CDR1. In an embodiment, the TCR that specifically binds to a mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR1, wherein the TRAV4 CDR1 comprises the following amino acid sequence: NNIATNDYI (SEQ ID NO: 81).

[0314] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR2. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR2, wherein the TRAV4 CDR2 comprises the following amino acid sequence: QGYKTKV (SEQ ID NO: 82).

[0315] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR3. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR3, wherein the TRAV4 CDR3 comprises the following amino acid sequence: CLVGDFNSNSGYALNF (SEQ ID NO: 83).

[0316] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV4 CDR1, a TRAV4 CDR2, and a TRAV4 CDR3.

[0317] In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV4. In an embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRAV4, wherein the variable domain of TRAV4 comprises the following amino acid sequence: LAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGD (SEQ ID NO: 175).

[0318] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 84).

[0319] In an embodiment, the TCR comprises a TCR alpha chain comprising the following amino acid sequence: MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDFNSNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 85).

[0320] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0321] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a T cell receptor beta variable region 7-2 (TRBV7-2) CDR1. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR1, wherein the TRBV7-2 CDR1 comprises the following amino acid sequence: ISGHTAL (SEQ ID NO: 86).

[0322] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR2, wherein the TRBV7-2 CDR2 comprises the following amino acid sequence: YFQGNSAP (SEQ ID NO: 87).

[0323] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR3. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR3, wherein the TRBV7-2 CDR3 comprises the following amino acid sequence: CASKVYGYTF (SEQ ID NO: 88).

[0324] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR1, a TRBV7-2 CDR2, and a TRBV7-2 CDR3.

[0325] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV7-2. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable domain of TRBV7-2, wherein the variable domain of TRBV7-2 comprises the following amino acid sequence: GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASK (SEQ ID NO: 176).

[0326] In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises a constant domain. In an embodiment, the TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises a constant domain, wherein the constant domain comprises the following amino acid sequence: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 89).

[0327] In an embodiment, the TCR comprises a TCR beta chain comprising the following amino acid sequence: MGTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASKVYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 90).

[0328] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3, and binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule. R GVGKSAL (SEQ ID NO: 15).

[0329] In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In an embodiment, the TCR comprises (a) a TCR a chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3, and binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule. R GVGKSAL (SEQ ID NO: 15).

[0330] In one embodiment, the TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises at least one of the CDRs selected from the group consisting of: TRAV4-CDR1 : SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1 : SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV4-CDR1 : SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1 : SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV4-CDR1 : SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1 : SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule. In one embodiment, the TCR comprises at least one of the CDRs selected from the group consisting of: TRAV4-CDR1 : SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1 : SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule, wherein the mRAS peptide comprises the sequence of GVGKSAL (SEQ ID NO: 15). R GVGKSAL (SEQ ID NO: 15).

[0331] In another embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises all of the CDRs from the group consisting of: TRAV4-CDR1: SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1: SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV4-CDR1: SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1: SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation at a position relative to RAS G12. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV4-CDR1: SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1: SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule. In one embodiment, the TCR comprises all of the CDRs from the group consisting of: TRAV4-CDR1: SEQ ID NO: 81; TRAV4-CDR2: SEQ ID NO: 82; TRAV4-CDR3: SEQ ID NO: 83; TRBV7-2-CDR1: SEQ ID NO: 86; TRBV7-2-CDR2: SEQ ID NO: 87; and TRBV7-2-CDR3: SEQ ID NO: 88, or one or more variants thereof, and binds to an mRAS peptide comprising a G12R mutation in the context of an HLA-B*07:02 molecule, wherein the mRAS peptide has the amino acid sequence of GVGKSAL (SEQ ID NO: 15). R GVGKSAL (SEQ ID NO: 15) in the context of an HLA-B*07:02 molecule.

[0332] In one embodiment, the composition comprises a fusion protein comprising a TCR alpha chain and a TCR beta chain as described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha chain from the TCR beta chain. In one embodiment, the linker domain is a cleavable linker domain. For example, in one embodiment, the linker domain comprises a GSG-T2A domain. In one embodiment, the GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 91).

[0333] In one embodiment, the composition comprises a fusion protein comprising an amino acid sequence of: MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDFNSNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGEGRGSLLTCGDVEENPGPMGTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASKVYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 92).

[0334] In certain embodiments, the composition comprises a fusion protein comprising a linker domain separating the TCR a chain and the TCR β chain. In one embodiment, the linker domain is a cleavable linker domain. Any suitable linker domain can be used such that the functionality of both the a chain and the β chain are preserved.

[0335] In certain embodiments, the compositions comprise a peptide or polypeptide (e.g., an mRAS peptide antigen or a TCR) comprising an amino acid sequence that is substantially homologous to an amino acid sequence of an mRAS peptide, a TCR, or a portion thereof described herein and retains the function of the original amino acid sequence. For example, in certain embodiments, the amino acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% degree of identity to the original amino acid sequence.

[0336] In some embodiments, the compositions comprise a peptide having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations (such as point mutations) relative to an amino acid sequence of an mRAS peptide or a TCR, or a portion thereof described herein.

[0337] In some embodiments, the TCR comprises an amino acid sequence having at least about 85% amino acid identity to one or more of the CDR sequences described herein. The present application encompasses TCRs having CDR sequences that are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identical to the CDR sequences described herein.

[0338] In one embodiment, the compositions comprise a polypeptide having CDR sequences that are at least about 85% identical to the CDR sequences described herein. The present application encompasses polypeptides having CDR sequences that are at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identical to the CDR sequences described herein.

[0339] Peptides of the present application can be prepared using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge JY et al. (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A peptide synthesizer (Perkin Elmer) according to the manufacturer’s instructions. Alternatively, the peptides can be prepared recombinantly or by cleavage from longer polypeptides. The composition of the peptides can be confirmed by amino acid analysis or sequencing.

[0340] A variant of a polypeptide according to the present application can be (i) a variant in which one or more amino acid residues are substituted by a conservative or non-conservative amino acid residue, preferably a conservative amino acid residue, and such substituted amino acid residues can or can not be amino acid residues encoded by the genetic code, (ii) a variant in which one or more modified amino acid residues are present (e.g., residues modified by attachment of a substituent group), (iii) a variant in which the polypeptide is an alternative splice variant of a polypeptide of the present application, (iv) a fragment of the polypeptide and / or (v) a variant in which the polypeptide is fused to another polypeptide, such as a leader or secretion sequence or a sequence for purification (e.g., His-tag) or for detection (e.g., S v5 epitope tag). Fragments include polypeptides produced by proteolytic cleavage of the original sequence, including multiple site proteolysis. Variants can be post-translationally or chemically modified. Such variants are considered to be within the scope of one skilled in the art according to the teachings herein.

[0341] As is known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide, and conservative amino acid substitutions thereof, to the sequence of a second polypeptide. Variants are defined to include polypeptide sequences that differ from the original sequence (preferably differ from the original sequence by less than 40% of the residues in each segment of interest, more preferably differ from the original sequence by less than 25% of the residues in each segment of interest, more preferably differ from the original sequence by less than 10% of the residues in each segment of interest, most preferably differ from the original protein sequence by only a few residues in each segment of interest) and at the same time are sufficiently homologous to the original sequence to maintain the ability of the original sequence to function and / or bind ubiquitin or ubiquitinated proteins. The present application includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides is determined using computer algorithms and methods well known to those of skill in the art. Identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215:403-410 (1990)].

[0342] The polypeptides of the present application can be post-translationally modified. For example, post-translational modifications falling within the scope of the present application include signal peptide cleavage, glycosylation, acetylation, prenylation, proteolysis, myristoylation, protein folding, and proteolytic processing, among others. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding dog microsomal membranes or Xenopus oocyte extracts (U.S. Patent No. 6,103,489) to standard translation reactions.

[0343] The polypeptides of the present application can include unnatural amino acids formed by post-translational modification or by the introduction of unnatural amino acids during translation. A variety of methods are available for the introduction of unnatural amino acids during protein translation. For example, special tRNAs, such as tRNAs with suppressor properties, suppressor tRNAs, have been used in the process of site- specific unnatural amino acid replacement (SNAAR). In SNAAR, a unique codon is required on the mRNA and suppressor tRNA, which is used to target an unnatural amino acid to a unique site during protein synthesis (described in WO 90 / 05785). However, suppressor tRNAs are not necessarily recognized by aminoacyl tRNA synthetases present in protein translation systems. In some cases, an unnatural amino acid can be formed after aminoacylation of a tRNA molecule using a chemical reaction that specifically modifies a natural amino acid and does not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications. For example, the epsilon-amino group of lysine attached to its cognate tRNA (tRNA LYS ) can be modified with an amine-specific photoaffinity label.

[0344] The peptides of the present application can be converted into pharmaceutically acceptable salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like; or with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluene sulfonic acid.

[0345] Figure 24

[0346] In one aspect, the present application provides compositions comprising an isolated nucleic acid molecule encoding one or more of the peptides or polypeptides described herein. For example, in certain aspects, the compositions comprise DNA, RNA, mRNA, or cDNA encoding one or more of the peptides or polypeptides described herein.

[0347] In one embodiment, the composition comprises one or more isolated nucleic acid molecules encoding one or more antigenic mRAS peptides described herein. For example, in one embodiment, the composition comprises one or more isolated nucleic acid molecules encoding one or more antigenic mRAS peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16.

[0348] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-92. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that is substantially homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-92. For example, in certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-92. In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence that has one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations, such as point mutations, relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-92.

[0349] In one embodiment, the composition comprises one or more isolated nucleic acid molecules encoding one or more TCRs described herein, one or more CDRs described herein, one or more alpha chains described herein, one or more beta domains described herein, one or more variable domains described herein, one or more constant domains described herein, one or more linkers described herein, or one or more fusion proteins described herein.

[0350] Nucleic acid molecules encoding TCR 831

[0351] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV39 CDR1, a TRAV39 CDR2, and a TRAV39 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV39 CDR1 comprising an amino acid sequence of SEQ ID NO: 17, a TRAV39 CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a TRAV39 CDR3 comprising an amino acid sequence of SEQ ID NO: 19. In one embodiment, the nucleic acid sequence encoding the TRAV39 CDR1 comprises ACCACTTCAGA (SEQ ID NO: 93). In one embodiment, the nucleic acid sequence encoding the TRAV39 CDR2 comprises TTGCTATCAAATGGAGCAGTG (SEQ ID NO: 94). In one embodiment, the nucleic acid sequence encoding the TRAV39 CDR3 comprises GCCGTGGACAAGGATGGGGGTTACC (SEQ ID NO: 95).

[0352] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a variable domain of TRAV39, the variable domain comprising an amino acid sequence of SEQ ID NO: 20. In one embodiment, the nucleic acid sequence encoding the TRAV39 variable domain comprises: ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGACAGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGATTAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCACATCACAGCTGCCGTGCATGACCTCTCTGCCACCTACTTCTGTGCCGTGGACAAGGATGGGGGTTACCAGAAAGTTACCTTTGGAACTGGAACAAAGCTCCAAGTCATCCCAA (SEQ ID NO: 96).

[0353] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 97).

[0354] In an embodiment, the nucleic acid molecule encodes a TCR a chain comprising the amino acid sequence of SEQ ID NO: 22. In an embodiment, the nucleic acid sequence encoding the TCR a chain comprises: ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGACAGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGATTAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCACATCACAGCTGCCGTGCATGACCTCTCTGCCACCTACTTCTGTGCCGTGGACAAGGATGGGGGTTACCAGAAAGTTACCTTTGGAACTGGAACAAAGCTCCAAGTCATCCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 98).

[0355] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV20-1 CDR1 comprising the amino acid sequence of SEQ ID NO: 23, TRBV20-1 CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and TRBV20-1 CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the nucleic acid sequence encoding TRBV20-1 CDR1 comprises GACTTTCAGGCCACAACT (SEQ ID NO: 99). In one embodiment, the nucleic acid sequence encoding TRBV20-1 CDR2 comprises TCCAATGAGGGCTCCAAGGCC (SEQ ID NO: 100). In one embodiment, the nucleic acid sequence encoding TRBV20-1 CDR3 comprises AGTGCTAGCCCACGGGCGGGACAGTTGAGCTCCTATAATTCACCCCTCCAC (SEQ ID NO: 101).

[0356] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a variable domain comprising the amino acid sequence of SEQ ID NO: 26. In one embodiment, the nucleic acid sequence encoding the TRBV20-1 variable domain comprises: ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTACCTGGGAGCTTGGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGCCCACGGGCGGGACAGTTGAGCTCCTATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGAC (SEQ ID NO: 102).

[0357] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC (SEQ ID NO: 103).

[0358]

[0359] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 105).

[0360]

[0361] A nucleic acid molecule encoding TCR 833

[0362] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV12-1 CDR1 comprising the amino acid sequence of SEQ ID NO:32, TRAV12-1 CDR2 comprising the amino acid sequence of SEQ ID NO:32, and TRAV12-1 CDR3 comprising the amino acid sequence of SEQ ID NO:33. In one embodiment, the nucleic acid sequence encoding TRAV12-1 CDR1 comprises AACAGTGCTTCTCAGTCT (SEQ ID NO: 107). In one embodiment, the nucleic acid sequence encoding TRAV12-1 CDR2 comprises GTATACTCCAGTGGTAAC (SEQ ID NO: 108). In one embodiment, the nucleic acid sequence encoding TRAV12-1 CDR3 comprises GCGGTGAACCCCCCGGACACAGGCTTTCAGAAACTTGTA (SEQ ID NO: 109).

[0363] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a variable domain comprising the amino acid sequence of SEQ ID NO: 34. In an embodiment, the nucleic acid sequence encoding the TRAV12-1 variable domain comprises: ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAACGAAGATGGAAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGCGGTGAACCCCCCGGACACAGGCTTTCAGAAACTTGTATTTGGAACTGGCACCCGACTTCTGGTCAGTCCAA (SEQ ID NO: 110).

[0364] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 35. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 111).

[0365] In an embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 36. In an embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises: ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAACGAAGATGGAAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGCGGTGAACCCCCCGGACACAGGCTTTCAGAAACTTGTATTTGGAACTGGCACCCGACTTCTGGTCAGTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 112).

[0366] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: a TRBV28 CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a TRBV28 CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and a TRBV28 CDR3 comprising an amino acid sequence of SEQ ID NO: 39. In one embodiment, the nucleic acid sequence encoding the TRBV28 CDR1 comprises ATGGACCATGAAAAT (SEQ ID NO: 113). In one embodiment, the nucleic acid sequence encoding the TRBV28 CDR2 comprises TCATATGATGTTAAAATG (SEQ ID NO: 114). In one embodiment, the nucleic acid sequence encoding the TRBV28 CDR3 comprises GCCAGCAGTTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTAC (SEQ ID NO: 115).

[0367] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a variable domain of TRBV28, the variable domain comprising an amino acid sequence of SEQ ID NO: 40. In one embodiment, the nucleic acid sequence encoding the TRBV28 variable domain comprises: ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCCAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA (SEQ ID NO: 116).

[0368] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 41. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC (SEQ ID NO: 117).

[0369] In one embodiment, the nucleic acid molecule encodes a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 42.In one embodiment, the nucleic acid sequence encoding the TCR beta chain comprises: ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCCAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 118).

[0370] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 43. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 119).

[0371]

[0372] A nucleic acid molecule encoding a TCR 897

[0373] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV17 CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a TRAV17 CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a TRAV17 CDR3 comprising the amino acid sequence of SEQ ID NO: 47. In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR1 comprises ACTAGTATAAACAAT (SEQ ID NO: 121). In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR2 comprises ATACGTTCAAATGAAAGAGAG (SEQ ID NO: 122). In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR3 comprises TGTGCTACGGACCCTGGAGGCTTCAAAACTATCTTT (SEQ ID NO: 123).

[0374] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 48. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 124).

[0375] In an embodiment, the nucleic acid molecule encodes a TCR a chain comprising the amino acid sequence of SEQ ID NO: 49. In an embodiment, the nucleic acid sequence encoding the TCR a chain comprises: ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGTTCCTTGTTGATCACGGCTTCCCGGGCAGCAGACACTGCTTCTTACTTCTGTGCTACGGACCCTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 125).

[0376] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV11-2 CDR1 comprising the amino acid sequence of SEQ ID NO:50, TRBV11-2 CDR2 comprising the amino acid sequence of SEQ ID NO:51, and TRBV11-2 CDR3 comprising the amino acid sequence of SEQ ID NO:52. In one embodiment, the nucleic acid sequence encoding TRBV11-2 CDR1 comprises TCTGGCCATGCTACC (SEQ ID NO:126). In one embodiment, the nucleic acid sequence encoding TRBV11-2 CDR2 comprises TTTCAGAATAACGGTGTA (SEQ ID NO:127). In one embodiment, the nucleic acid sequence encoding TRBV11-2 CDR3 comprises TGTGCCAGCAGCTTATATGGGGGGTCGATCTCCTACGAGCAGTACTTC (SEQ ID NO:128).

[0377] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC (SEQ ID NO: 129).

[0378] In one embodiment, the nucleic acid molecule encodes a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 54.In one embodiment, the nucleic acid sequence encoding the TCR beta chain comprises: ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGAAGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCTGATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACTCCACTCTCAAGATCCAGCCTGCAAAGCTTGAGGACTCGGCCGTGTATCTCTGTGCCAGCAGCTTATATGGGGGGTCGATCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 130).

[0379] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 55. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 131).

[0380]

[0381] A nucleic acid molecule encoding TCR 896

[0382] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV19 CDR1 comprising the amino acid sequence of SEQ ID NO:57, TRAV19 CDR2 comprising the amino acid sequence of SEQ ID NO:58, and TRAV19 CDR3 comprising the amino acid sequence of SEQ ID NO:59. In one embodiment, the nucleic acid sequence encoding TRAV19 CDR1 comprises ACCCGTGATACTACTTATTAC (SEQ ID NO: 133). In one embodiment, the nucleic acid sequence encoding TRAV19 CDR2 comprises CGGAACTCTTTTGATGAGCAAAAT (SEQ ID NO: 134). In one embodiment, the nucleic acid sequence encoding TRAV19 CDR3 comprises TGTGCTCTGAGTGAGGCAGGAACCTACAAATACATCTTT (SEQ ID NO: 135).

[0383] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 60. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 136).

[0384] In an embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 61. In an embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises: ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGAGTGAGGCAGGAACCTACAAATACATCTTTGGAACAGGCACCAGGCTGAAGGTATTAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 137).

[0385] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: a TRBV9 CDR1, a TRBV9 CDR2, and a TRBV9 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: a TRBV9 CDR1 comprising an amino acid sequence of SEQ ID NO:62, a TRBV9 CDR2 comprising an amino acid sequence of SEQ ID NO:63, and a TRBV9 CDR3 comprising an amino acid sequence of SEQ ID NO:64. In one embodiment, the nucleic acid sequence encoding the TRBV9 CDR1 comprises TCTGGAGACCTCTCT (SEQ ID NO: 138). In one embodiment, the nucleic acid sequence encoding the TRBV9 CDR2 comprises CGGAACTCTTTTGATGAGCAAAAT (SEQ ID NO: 139). In one embodiment, the nucleic acid sequence encoding the TRBV9 CDR3 comprises TGTGCTCTGAGTGAGGCAGGAACCTACAAATACATCTTT (SEQ ID NO: 140).

[0386] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 65. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC (SEQ ID NO: 141).

[0387] In one embodiment, the nucleic acid molecule encodes a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 66.In one embodiment, the nucleic acid sequence encoding the TCR beta chain comprises: ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGCTGGGGGGGGACAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 142).

[0388] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 67. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 143).

[0389]

[0390] A nucleic acid molecule encoding a TCR 847

[0391] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: a TRAV17 CDR1 comprising the amino acid sequence of SEQ ID NO: 69, a TRAV17 CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and a TRAV17 CDR3 comprising the amino acid sequence of SEQ ID NO: 71. In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR1 comprises ACTAGTATAAACAAT (SEQ ID NO: 145). In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR2 comprises ATACGTTCAAATGAAAGAGAG (SEQ ID NO: 146). In one embodiment, the nucleic acid sequence encoding the TRAV17 CDR3 comprises GCTACTTTTCCTAACTTTGGAAATGAGAAATTAACC (SEQ ID NO: 147).

[0392] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 72. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 148).

[0393] In an embodiment, the nucleic acid molecule encodes a TCR a chain comprising the amino acid sequence of SEQ ID NO: 73. In an embodiment, the nucleic acid sequence encoding the TCR a chain comprises: ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGTTCCTTGTTGATCACGGCTTCCCGGGCAGCAGACACTGCTTCTTACTTCTGTGCTACTTTTCCTAACTTTGGAAATGAGAAATTAACCTTTGGGACTGGAACAAGACTCACCATCATACCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 149).

[0394] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV10-3 CDR1 comprising the amino acid sequence of SEQ ID NO:74, TRBV10-3 CDR2 comprising the amino acid sequence of SEQ ID NO:75, and TRBV10-3 CDR3 comprising the amino acid sequence of SEQ ID NO:76. In one embodiment, the nucleic acid sequence encoding TRBV10-3 CDR1 comprises GAGAACCACCGCTA (SEQ ID NO: 150). In one embodiment, the nucleic acid sequence encoding TRBV10-3 CDR2 comprises TCATATGGTGTTAAAGAT (SEQ ID NO: 151). In one embodiment, the nucleic acid sequence encoding TRBV10-3 CDR3 comprises GCCATCAGTGAGTCGGAGCGGTACTACGAGCAGTAC (SEQ ID NO: 152).

[0395] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC (SEQ ID NO: 153).

[0396] In one embodiment, the nucleic acid molecule encodes a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 78.In one embodiment, the nucleic acid sequence encoding the TCR beta chain comprises: ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCCAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTTCTGTGCCATCAGTGAGTCGGAGCGGTACTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 154).

[0397] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 79. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 155).

[0398]

[0399] A nucleic acid molecule encoding TCR 864

[0400] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of: TRAV4 CDR1 comprising the amino acid sequence of SEQ ID NO: 81, TRAV4 CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and TRAV4 CDR3 comprising the amino acid sequence of SEQ ID NO: 83. In one embodiment, the nucleic acid sequence encoding TRAV4 CDR1 comprises AACATTGCTACAAATGATTAT (SEQ ID NO: 157). In one embodiment, the nucleic acid sequence encoding TRAV4 CDR2 comprises GGATACAAGACAAAA (SEQ ID NO: 158). In one embodiment, the nucleic acid sequence encoding TRAV4 CDR3 comprises CTCGTGGGTGACTTCAACTCAAATTCCGGGTATGCACTCAAC (SEQ ID NO: 159).

[0401] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 84. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 160).

[0402] In an embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 85. In an embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises: ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTGAGCCTTGCTAAGACCACCCAGCCCATCTCCATGGACTCATATGAAGGACAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGATTATATCACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATTATTCAAGGATACAAGACAAAAGTTACAAACGAAGTGGCCTCCCTGTTTATCCCTGCCGACAGAAAGTCCAGCACTCTGAGCCTGCCCCGGGTTTCCCTGAGCGACACTGCTGTGTACTACTGCCTCGTGGGTGACTTCAACTCAAATTCCGGGTATGCACTCAACTTCGGCAAAGGCACCTCGCTGTTGGTCACACCCCATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 161).

[0403] In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In embodiments, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising one or more of: TRBV7-2 CDR1 comprising the amino acid sequence of SEQ ID NO: 86, TRBV7-2 CDR2 comprising the amino acid sequence of SEQ ID NO: 87, and TRBV7-2 CDR3 comprising the amino acid sequence of SEQ ID NO: 88. In one embodiment, the nucleic acid sequence encoding TRBV7-2 CDR1 comprises TCAGGTCATACTGCC (SEQ ID NO: 162). In one embodiment, the nucleic acid sequence encoding TRBV7-2 CDR2 comprises TTCCAAGGCAACAGTGCA (SEQ ID NO: 163). In one embodiment, the nucleic acid sequence encoding TRBV7-2 CDR3 comprises GCCAGCAAGGTCTATGGCTACACC (SEQ ID NO: 164).

[0404] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence of SEQ ID NO: 89. In one embodiment, the nucleic acid sequence encoding the constant domain comprises: GAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC (SEQ ID NO: 165).

[0405] In one embodiment, the nucleic acid molecule encodes a TCR beta chain comprising the amino acid sequence of SEQ ID NO: 90.In one embodiment, the nucleic acid sequence encoding the TCR beta chain comprises: ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCATATCACACAGGAGCTGGAGTCTCCCAGTCCCCCAGTAACAAGGTCACAGAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGCCCTTTACTGGTACCGACAGAGGCTGGGGCAGGGCCTGGAGTTTTTAATTTACTTCCAAGGCAACAGTGCACCAGACAAATCAGGGCTGCCCAGTGATCGCTTCTCTGCAGAGAGGACTGGGGAATCCGTCTCCACTCTGACGATCCAGCGCACACAGCAGGAGGACTCGGCCGTGTATCTCTGTGCCAGCAAGGTCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA (SEQ ID NO: 166).

[0406] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR a chain and a TCR β chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR a chain and the TCR β chain. In one embodiment, the nucleic acid molecule encodes a GSG-T2A linker domain comprising the amino acid sequence of SEQ ID NO: 91. In one embodiment, the nucleic acid sequence encoding the GSG-T2A linker domain comprises: GGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 167).

[0407]

[0408] In certain embodiments, the nucleic acid sequence encoding the alpha chain constant region or the beta chain constant region of the TCR comprises a nucleic acid sequence that is resistant to gene editing, such as CRISPR-mediated gene editing.

[0409] Further, the present application encompasses isolated nucleic acids encoding an amino acid sequence having substantial identity to an amino acid sequence disclosed herein. In certain embodiments, the isolated nucleic acid sequence encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence disclosed herein.

[0410] Further, the present application encompasses isolated nucleic acids having substantial identity to a nucleic acid sequence disclosed herein. In certain embodiments, the isolated nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence disclosed herein.

[0411] The isolated nucleic acid sequence encoding a polypeptide of the present application can be obtained using any of a number of recombinant procedures known in the art, such as, for example, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0412] The isolated nucleic acid can comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including, for example, an isolated cDNA molecule or a functional fragment thereof, encoding a polypeptide of the present application. In one embodiment, the composition comprises an isolated RNA molecule or a functional fragment thereof, encoding a polypeptide of the present application.

[0413] The nucleic acid molecules of the present application can be modified to improve stability in serum or in growth media used for cell culture. Modifications can be added to enhance stability, functionality, and / or specificity and to minimize the immunostimulatory properties of the nucleic acid molecules of the present application. For example, to enhance stability, the 3’-residues can be stabilized against degradation, for example, they can be chosen such that they consist of purine nucleotides, in particular adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, for example, substitution of uridines with 2’-deoxythymidines is tolerated and does not affect the function of the molecule.

[0414] In one embodiment of the present application, the nucleic acid molecule can contain at least one modified nucleotide analog. For example, the ends can be stabilized by incorporation of modified nucleotide analogs.

[0415] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., including modifications to the phospho-sugar backbone). For example, the phosphodiester linkage of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphate group linking adjacent ribonucleotides is replaced by a modified group, such as a phosphorothioate group. In preferred sugar-modified ribonucleotides, the 2’ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is a C1-C6 alkyl, alkenyl, or alkynyl group and halo is F, Cl, Br, or I.

[0416] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The bases can be modified to block the activity of adenosine deaminases. Exemplary modified nucleobases include, but are not limited to, 5-position modified uridines and / or cytidines, such as 5-(2-amino)propyl uridine, 5-bromouridine; 8-position modified adenosines and / or guanosines, such as 8-bromoguanosine; deazanucleotides, such as 7-deazoadenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications can be combined.

[0417] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2’-H, 2’-O-methyl, or 2’-OH modification of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present application can have increased nuclease resistance. To increase nuclease resistance, the nucleic acid molecule can include, for example, 2’-modified ribose units and / or phosphorothioate linkages. For example, the 2’ hydroxyl (OH) group can be modified or replaced by a number of different “oxy” or “deoxy” substituents. To increase nuclease resistance, the nucleic acid molecules of the present application can include 2’-O-methyl, 2’-fluoro, 2’-O-methoxyethyl, 2’-O-aminopropyl, 2’-amino, and / or phosphorothioate linkages. The inclusion of locked nucleic acids (LNAs), ethylene nucleic acids (ENAs) (e.g., 2’-4’-ethylene-bridged nucleic acids), and certain nucleobase modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications) can also increase binding affinity to a target.

[0418] In one embodiment, the nucleic acid molecule comprises 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0- aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0- dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0- DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-0-methyl modification.

[0419] The present application also includes vectors into which a nucleic acid of the present application has been inserted. There are a number of suitable vectors for use in the present application.

[0420] Briefly, expression of a natural or synthetic nucleic acid encoding a peptide of the present application is typically achieved by operably linking the nucleic acid encoding the peptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of expression of the desired nucleic acid sequence.

[0421] Vectors of the present application can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the present application provides a gene therapy vector.

[0422] The isolated nucleic acids of the present application can be cloned into a number of types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0423] Furthermore, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0424] A number of viral-based systems have been developed for the transfer of genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. A number of adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0425] For example, vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from onco-retroviruses such as murine leukemia virus, as they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of being less immunogenic. In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have emerged as a powerful gene delivery tool for the treatment of various disorders. AAV vectors possess a number of features that make them highly suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and efficient manner. By selecting the appropriate combination of AAV serotype, promoter, and method of delivery, expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells.

[0426] In certain embodiments, the vector further comprises conventional control elements that are operably linked to the transgene in a manner that permits transcription, translation, and / or expression of the transgene in a cell transfected with the plasmid vector or infected with a virus produced by the application. As used herein, "operably linked" sequences include expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences, including promoters that are natural, constitutive, inducible and / or tissue-specific, are known in the art and can be utilized.

[0427] Additional promoter elements (e.g., enhancers) modulate the frequency of transcription initiation. Generally, these elements are located in the region 30-110 bp upstream of the initiation site, but it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, so that the promoter function is preserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to drop. Depending on the promoter, individual elements can appear to act synergistically or independently to activate transcription.

[0428] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor 1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, the present application should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present application. The use of inducible promoters provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked thereto when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0429] Enhancer sequences found on vectors also modulate the expression of genes contained therein. Generally, enhancers bind to protein factors to enhance transcription of a gene. Enhancers can be located upstream or downstream of the genes that they regulate. Enhancers can also be tissue specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present application comprise one or more enhancers to increase transcription of genes present in the vector.

[0430] To assess expression of the peptides, the expression vectors to be introduced into cells can also comprise a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vectors. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked on both sides by appropriate regulatory sequences to enable their expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0431] Reporter genes are used to identify potential transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue, and it encodes a polypeptide whose expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is measured at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or commercially available. Generally, constructs with minimal 5' flanking regions that exhibit the highest expression levels of the reporter gene are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of drugs to regulate promoter-driven transcription.

[0432] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) using any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0433] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0434] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors (and especially retroviral vectors) have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0435] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems (such as macromolecular complexes, nanocapsules, microspheres, beads) and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery carrier in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0436] In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. It is contemplated that a nucleic acid will be introduced into a host cell (in vitro, ex vivo, or in vivo) using a lipid formulation. In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome by a linking molecule that associates with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector related compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in "collapsed" structures. They can also simply be dispersed in solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include the fat droplets naturally occurring in cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0437] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidyl choline ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidyl glycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vehicles formed by closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-organize into a bilayer structure with the water and dissolved solutes trapped between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicular structures in solution are also contemplated. For example, the lipids can take a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Cationic liposome-nucleic acid complexes are also contemplated.

[0438] Regardless of the method used to introduce foreign nucleic acid into a host cell, to confirm the presence of a recombinant DNA sequence in a host cell, a variety of assays can be performed. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of the application.

[0439] In one embodiment, the isolated nucleic acid encoding a polypeptide of the application comprises in vitro transcribed (IVT) RNA. RNA is produced by in vitro transcription using a template produced by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted to a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase by PCR. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other appropriate source of DNA.

[0440] In one embodiment, the DNA used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence in the genome of an organism. In one embodiment, the DNA is a full-length gene of interest or a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene including 5' and 3' UTRs. Alternatively, the DNA can be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a DNA sequence comprising portions of genes linked together to form an open reading frame encoding a fusion protein. The portions of DNA linked together can be from a single organism or from more than one organism.

[0441] Genes that can be used as a source of DNA for PCR include genes that encode polypeptides that provide a therapeutic or prophylactic effect to an organism or that can be used to diagnose a disease or disorder in an organism. Preferred genes are genes that are useful for short-term therapy, or genes for which there are safety concerns with respect to dosing or expression of the gene. For example, to treat cancer, an autoimmune disorder, a parasitic, viral, bacterial, fungal or other infection, the transgene to be expressed can encode a polypeptide that functions as a ligand or receptor for a cell of the immune system or that can function to stimulate or inhibit the immune system of the organism. In some embodiments, it is not desirable to have a long-term, sustained stimulation of the immune system, nor is it necessary to have a lasting change after successful treatment, as this can trigger new problems. For treatment of autoimmune disorders, it can be desirable to suppress or dampen the immune system during the episode, but not long-term, which can result in the patient becoming overly sensitive to infection.

[0442] PCR is used to generate templates for in vitro transcription of mRNA for transfection. Methods for performing PCR are well known in the art. Primers for PCR are designed to have regions that are substantially complementary to regions of the DNA used as a template for PCR. As used herein, "substantially complementary" refers to nucleotide sequences in which most or all of the bases are complementary or one or more bases are not complementary or are mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of genes that are typically transcribed in cells (open reading frames), including 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are produced by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that is substantially complementary to nucleotides on a DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5' position of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that is substantially complementary to nucleotides of a double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3' position of the DNA sequence to be amplified relative to the coding strand.

[0443] Any DNA polymerase that can be used for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from many sources.

[0444] Chemical structures that can promote stability and / or translational efficiency can also be used. The RNA preferably has a 5' and 3' UTR. In one embodiment, the 5' UTR is between 0 and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences that can be added to the coding region can be varied by different methods including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR length required to achieve optimal translational efficiency after transfection of the transcribed RNA.

[0445] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are non-endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are non-endogenous to the gene of interest can be used to modify the stability and / or translational efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to decrease the stability of mRNA. Thus, the 3' UTR can be selected or designed based on the properties of the UTR well known in the art to increase the stability of the transcribed RNA.

[0446] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is non-endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the translational efficiency of some RNA transcripts, but it does not appear to be required for all RNAs to achieve efficient translation. The requirement of many mRNAs for a Kozak sequence is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0447] To enable the synthesis of RNA from a DNA template without the need for gene cloning, a transcriptional promoter should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0448] In preferred embodiments, the mRNA has a cap at the 5' end and a poly(A) tail at the 3' end, which determines ribosome binding, initiation of translation, and stability of the mRNA in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of linearized plasmid DNA at the end of the 3'UTR produces a normal-sized mRNA, which is not efficient in eukaryotic transfection, even if it is linearized after transcription.

[0449] On a linear DNA template, the bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0450] The traditional method of incorporating a poly A / T extension into a DNA template is molecular cloning. However, the incorporation of a poly A / T sequence into a plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning procedure not only laborious and time-consuming, but often unreliable. This is why there is a great need for a method to construct a DNA template with a poly A / T 3' extension without the need for cloning.

[0451] The poly A / T segment of the transcriptional DNA template can be generated during PCR by using a reverse primer containing a poly T tail (such as a 100 T tail (size can be 50-5000 T)), or after PCR by any other method including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces their degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is increased from 100 nucleotides to between 300 and 400 nucleotides, resulting in about a two-fold increase in RNA translation efficiency. Additionally, the attachment of different chemical groups at the 3' end can increase the stability of the mRNA. Such attachment can contain modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase RNA stability.

[0452] The poly(A) tail of the RNA can be further extended in vitro after transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, the length of the poly(A) tail is increased from 100 nucleotides to between 300 and 400 nucleotides, resulting in about a two-fold increase in RNA translation efficiency. Additionally, the attachment of different chemical groups at the 3' end can increase the stability of the mRNA. Such attachment can contain modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further increase RNA stability.

[0453] 5' caps also provide stability to RNA molecules. In preferred embodiments, the RNA produced by the methods disclosed herein include a 5' cap. Techniques known in the art and described herein are used to provide 5' caps (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7: 1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0454] The RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates ribosome binding to the mRNA independent of a cap and facilitates the initiation of translation. Any solutes suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0455] The RNA can be introduced into the target cell using any of a variety of different methods (e.g., commercially available methods), including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Mass) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or bioparticle delivery systems such as "gene guns" (see, e.g., Nishikawa et al. Hum Gene Ther., 12(8):861-70 (2001).

[0456] In another aspect, RNA constructs can be delivered into cells by electroporation. See, e.g., formulations and methods for electroporating nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630 Al, US 2005 / 0070841 Al, US 2004 / 0059285 Al, US 2004 / 0092907 Al. Various parameters required for electroporation of any known cell type, including electric field strength, are generally known in the relevant research literature as well as in many patents and applications in this field. See, e.g., as described in U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264, and U.S. Patent No. 7,173,116. Devices for electroporation therapy applications are commercially available, e.g., MedPulser TM DNA electroporation therapy system (Inovio / Genetronics, San Diego, Calif.), and described in patents such as U.S. Patent No. 6,567,694; U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701, and U.S. Patent No. 6,233,482; electroporation can also be used to transfect cells in vitro, as described in, e.g., US20070128708 Al. Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, into cells using any of the many available devices and electroporation systems known to those skilled in the art, provides an exciting new means of delivering RNAs of interest into target cells.

[0457] Figure 25

[0458] In certain embodiments, the compositions of the application comprise cells modified to comprise or express a peptide of the application. In certain embodiments, the cells are genetically modified by contacting the cells with an isolated nucleic acid encoding a polypeptide described herein, such as an mRAS peptide, a TCR, or a fusion protein comprising a TCR alpha chain and a TCR beta chain.

[0459] In some embodiments, nucleic acid sequences are delivered into cells using retroviral or lentiviral vectors. For example, retroviral and lentiviral vectors expressing peptides of the application can be delivered into different types of eukaryotic cells as well as tissues and whole organisms using transduced cells as carriers or cell-free local or systemic delivery of encapsulated, conjugated, or naked vectors. The methods used can be used for any purpose for which stable expression is desired or sufficient.

[0460] In other embodiments, nucleic acid sequences are delivered into cells using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered into different types of eukaryotic cells as well as tissues and whole organisms using transfected cells as vehicles or cell-free local or systemic delivery of encapsulated, conjugated or naked mRNA for any purpose for which transient expression is desired or sufficient.

[0461] In certain embodiments, the cell can be any suitable cell type that can express a desired peptide. In certain embodiments, the modified cell is used in a method of introducing the cell into a recipient. In certain embodiments, the cell is autologous, allogeneic, syngeneic, or xenogeneic to the recipient. In certain embodiments, the cell is derived from a stem cell or a precursor cell. In some embodiments, the stem cell or precursor cell from which the modified cell is derived is autologous, allogeneic, syngeneic, or xenogeneic to the recipient.

[0462] In one embodiment, the cell is an immune cell. For example, in certain embodiments, the composition comprises an immune cell that comprises or expresses one or more mRAS peptides or TCRs described herein. Exemplary immune cells that can comprise or express one or more TCRs described herein include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, and gamma delta T cells), natural killer (NK) cells, and NK T cells. Exemplary immune cells that can comprise or express one or more mRAS peptides described herein include, but are not limited to, antigen presenting cells, dendritic cells, B cells, macrophages, Langerhans cells, T cells, NK cells, NK T cells. Exemplary immune cells include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, and gamma delta T cells), B cells, antigen presenting cells (APCs), natural killer (NK) cells, and NK T cells.

[0463] In one embodiment, the cell is an antigen presenting cell (APC). For example, in certain embodiments, the composition comprises an APC modified to comprise or express a mRAS peptide described herein. Exemplary APCs include, but are not limited to, dendritic cells (DCs), macrophages, Langerhans cells, B cells, and the like.

[0464] The disclosed compositions and methods can be applied to modulate T cell activity in basic research and therapeutics, in the field of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill target cancer cells.

[0465] Prior to expansion and genetic modification of the T cells of the application, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the application, any number of T cell lines available in the art can be used. In certain embodiments of the application, T cells can be obtained from a unit of blood collected from a subject using any technique known to one of skill in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction and place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the application, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, and can lack magnesium, or can lack many, if not all, divalent cations. Again, surprisingly, the initial activation step in the absence of calcium results in amplified activation. As will be readily appreciated by one of ordinary skill in the art, the washing step can be accomplished by methods known to one of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers (e.g., Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffers). Alternatively, unwanted components can be removed from the apheresis sample and the cells resuspended directly in culture media.

[0466] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting mononuclear cells, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation. Specific T cell subpopulations can be further isolated by positive or negative selection techniques, such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +T cells. For example, in one implementation, by conjugating anti-CD3 / anti-CD28 (i.e., 3x28) beads (such as... T cells are isolated by incubating M-450 CD3 / CD28T cells together for a duration sufficient to positively select the desired T cells. In one embodiment, the incubation period is approximately 30 minutes. In another embodiment, the incubation period ranges from 30 minutes to 36 hours or longer, and includes all integer values ​​in between. In yet another embodiment, the incubation period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the incubation period is 10 to 24 hours. In one preferred embodiment, the incubation period is 24 hours. For isolating T cells from leukemia patients, using a longer incubation time (such as 24 hours) can increase cell yield. In any case where there are fewer T cells compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, a longer incubation time can be used to isolate T cells. Furthermore, using a longer incubation time can increase the capture efficiency of CD8+ T cells. Therefore, by simply shortening or lengthening the time allowed for T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T-cell ratio (as further described herein), selection can be preferentially performed on or against T-cell subsets at the start of culture or at other points during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, selection can be preferentially performed on or against T-cell subsets at the start of culture or at other desired points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of this invention. In some embodiments, it may be desirable to perform a selection procedure and use “unselected” cells during activation and expansion. “Unselected” cells may also undergo additional rounds of selection.

[0467] Enriching T cell populations by negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4 by negative selection... + Cellular monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some implementations, enrichment or positive selection may be required for cells that typically express CD4. + CD25 + CD62L hi GITR + and FoxP3 +regulatory T cells. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0468] To isolate the desired cell population by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it can be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In further embodiments, a concentration of greater than 1 billion cells / ml is used. In further embodiments, a cell concentration of 100 million, 150 million, 200 million, 250 million, 300 million, 350 million, 400 million, 450 million, or 500 million cells / ml is used. In yet further embodiments, a cell concentration of 750 million, 800 million, 850 million, 900 million, 950 million, or 1 billion cells / ml is used. In further embodiments, a concentration of 1.25 billion or 1.5 billion cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that can weakly express the target antigen of interest (such as CD28 negative T cells) or more efficient capture of cells from samples in which many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations can have therapeutic value and are desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8 + T cells that typically have weaker CD28 expression.

[0469] In related embodiments, it can be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), the interaction between the particles and the cells is minimized. This selects for cells that express high amounts of the desired antigen that bind to the particles. For example, CD4 + T cells express higher levels of CD28 and are more efficiently captured than the dilute concentration of CD8 + T cells. In one embodiment, the concentration of cells used is 5X10 6 / ml. In other embodiments, the concentration used can be from about 1X10 5 / ml to 1X10 6 / ml, and any integer value in between.

[0470] In other embodiments, the cells can be incubated on the rotors at different speeds for different lengths of time at 2-10°C or room temperature.

[0471] The T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent mononuclear cells from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, one approach involves the use of PBS containing 20% DMSO and 8% human serum albumin, or a media containing 10% Dextran 40 and 5% glucose, 20% human serum albumin, and 7.5% DMSO, or a media containing 31.25% Plasmalyte-A, 31.25% glucose 5%, 0.45% NaCl, 10% Dextran 40 and 5% glucose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media containing, e.g., hydroxyethyl starch and PlasmaLyte A, followed by freezing the cells at a rate of 1 ° per minute to -80 °C and storage in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can also be used in addition to immediate uncontrolled freezing at -20 °C or in liquid nitrogen.

[0472] In certain embodiments, the cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation using the methods of the application.

[0473] It is also contemplated in the context of the present application to collect a blood sample or an apheresis product from a subject at a time period prior to when expanded cells as described herein can be needed. Thus, a source of cells to be expanded can be collected at any point in time necessary and the desired cells (e.g., T cells) isolated and frozen for later use in a T cell therapy for any number of diseases or conditions that would benefit from a T cell therapy, such as those described herein. In one embodiment, the blood sample or apheresis sample is taken from a generally healthy subject. In certain embodiments, the blood sample or apheresis sample is taken from a generally healthy subject who is at risk for developing a disease, but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen and used at a later time. In certain embodiments, the sample is collected from a patient soon after diagnosis of a particular disease as described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis sample of a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, anti-viral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immune depleting agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70 S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In further embodiments, cells are isolated for a patient and the cells are frozen for later use in conjunction with (e.g., prior to, concurrently with, or following) a bone marrow or stem cell transplant, T cell ablative therapy using a chemotherapy agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to a B cell ablative therapy such as an agent that reacts with CD20 (e.g., Rituxan) and can be frozen for later use in a therapy following a B cell ablative therapy such as an agent that reacts with CD20 (e.g., Rituxan).

[0474] In additional embodiments of the application, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that, following treatment for certain cancers, particularly following treatment with drugs that compromise the immune system, the quality of the T cells obtained can be optimal or improved for their ability to expand ex vivo shortly after treatment during the period when the patient is generally recovering from treatment. Likewise, following ex vivo manipulation using the methods described herein, these cells can be in a preferred state for enhanced engraftment and expansion in vivo. Thus, it is contemplated in the context of the present application to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage during this recovery phase. Furthermore, in certain embodiments, mobilization (e.g., with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the re-population, recirculation, regeneration and / or expansion of particular cell types, particularly during a defined window of time after treatment. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0475] T cells can generally be activated and expanded using methods as described in, e.g., U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005, whether before or after the T cells are genetically modified to express the peptides of the application.

[0476] Generally, the T cells of the application are expanded by surface contact with agents that have attached thereto a signal related to stimulation of the CD3 / TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cell. Specifically, a population of T cells can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of a T cell surface with a costimulatory molecule, a ligand that binds the costimulatory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating proliferation of the T cells. For stimulation of CD4 + T cells or CD8 + T cells are expanded using an anti-CD3 antibody and an anti-CD28 antibody. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and 37228 (R&D Systems, Minneapolis, MN). The anti-CD3 antibody can be a monoclonal antibody, such as OKT3 (ATCC HB- 9568), or an antigen-binding fragment thereof. The anti-CD3 antibody can be immobilized on a surface, such as a plate or bead. The anti-CD3 antibody can be a monoclonal antibody, such as OKT3 (ATCC HB-9568), or an antigen-binding fragment thereof. The anti-CD3 antibody can be immobilized on a surface, such as a plate or bead. Other methods known in the art can also be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 1319 1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0477] In certain embodiments, the primary stimulation signal and the costimulation signal for T cells can be provided by different regimens. For example, the agents providing each signal can be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in "cis" format) or to separate surfaces (i.e., in "trans" format). Alternatively, one agent can be coupled to a surface while the other agent is in solution. In one embodiment, the agent providing the costimulation signal is bound to the surface of the cell, while the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then cross-linked to a surface (such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind to the agents). In this regard, see, e.g., U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810, with respect to artificial antigen presenting cells (aAPCs) contemplated for use in the present application for activating and expanding T cells.

[0478] In one embodiment, the two agents are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent providing the costimulation signal is an anti-CD28 antibody or antigen-binding fragment thereof; and the two agents are co-immobilized on the same bead in equal molecular amounts. In one embodiment, for CD4 +T cell expansion and T cell growth, using a 1 : 1 ratio of each antibody bound to the bead. In certain aspects of the application, a ratio of anti-CD3:CD28 antibodies bound to the bead is used such that an increase in T cell expansion is observed as compared to expansion observed using a 1 : 1 ratio. In a particular embodiment, an increase from about 1-fold to about 3-fold is observed as compared to expansion observed using a 1 : 1 ratio. In one embodiment, the ratio of anti-CD3:CD28 antibodies bound to the bead is in the range of 100: 1 to 1 : 100 and all integer values therebetween. In one aspect of the application, more anti-CD28 antibody is bound to the particle as compared to anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than 1. In certain embodiments of the application, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the bead is greater than 2: 1. In a particular embodiment, a 1 : 100 CD3:CD28 ratio of antibodies bound to the bead is used. In another embodiment, a 1 : 75 CD3:CD28 ratio of antibodies bound to the bead is used. In a further embodiment, a 1 : 50 CD3:CD28 ratio of antibodies bound to the bead is used. In another embodiment, a 1 : 30 CD3:CD28 ratio of antibodies bound to the bead is used. In a preferred embodiment, a 1 : 10 CD3:CD28 ratio of antibodies bound to the bead is used. In another embodiment, a 1 : 3 CD3:CD28 ratio of antibodies bound to the bead is used. In yet another embodiment, a 3 : 1 CD3:CD28 ratio of antibodies bound to the bead is used.

[0479] Particle to cell ratios from 1 :500 to 500: 1 and any integer value therebetween can be used to stimulate T cells or other target cells. As can be readily appreciated by one of ordinary skill in the art, the particle to cell ratio can depend on the size of the particles relative to the target cells. For example, small sized beads can bind only a few cells, while larger beads can bind many cells. In certain embodiments, the cell to particle ratio is in the range from 1 : 100 to 100: 1 and any integer value therebetween, and in further embodiments, the ratio includes 1 :9 to 9: 1 and any integer value therebetween, can also be used to stimulate T cells. As noted above, the ratio of anti-CD3- and anti-CD28-coupled particles to T cells resulting in T cell stimulation can vary, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1, with a preferred ratio of at least 1 : 1 particles per T cell. In one embodiment, a particle to cell ratio of 1 : 1 or less is used. In a particular embodiment, the preferred particle to cell ratio is 1 :5. In further embodiments, the particle to cell ratio can vary depending on the number of days of stimulation. For example, in one embodiment, the particle to cell ratio is from 1 : 1 to 10: 1 on the first day, and additional particles are added to the cells every day or every other day for up to 10 days, with a final ratio of 1 : 1 to 1 : 10 (based on cell count on the day of addition). In a particular embodiment, the particle to cell ratio is 1 : 1 on the first day of stimulation, and is adjusted to 1 :5 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, resulting in a final ratio of 1 : 1 on the first day of stimulation, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the particle to cell ratio is 2: 1 on the first day of stimulation, and is adjusted to 1 : 10 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, resulting in a final ratio of 1 : 1 on the first day of stimulation, and 1 : 10 on the third and fifth days of stimulation. Those of skill in the art will appreciate that a variety of other ratios can be suitable for use in the present application. In particular, the ratio will vary depending on the size of the particles and the size and type of the cells.

[0480] In further embodiments of the application, cells, such as T cells, are combined with the agent-coated beads, the beads and cells are then separated, and the cells are then cultured. In alternative embodiments, the agent-coated beads and cells are not separated prior to culture, but are cultured together. In further embodiments, the beads and cells are first concentrated by the application of force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0481] By way of example, cell surface proteins can be ligated by allowing paramagnetic beads with anti-CD3 and anti-CD28 attached (3x28 beads) to contact T cells. In one embodiment, cells (e.g., 10 4 to 10 9 cells) and beads (e.g., M-450 CD3 / CD28 T paramagnetic beads, at a ratio of 1 : 1) are combined in a buffer, preferably PBS (without divalent cations, such as calcium and magnesium). Again, one of ordinary skill in the art can readily appreciate that any cell concentration can be used. For example, target cells can be very rare in a sample, and only 0.01% of a sample or the entire sample (i.e., 100%) can contain target cells of interest. Thus, any number of cells is within the context of the present application. In certain embodiments, it can be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of the cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, greater than 100 million cells / ml is used. In further embodiments, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In further embodiments, a concentration of 125 million or 150 million cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that can weakly express target antigens of interest, such as CD28-negative T cells. In certain embodiments, such cell populations can have therapeutic value and are desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that typically have weaker CD28 expression.

[0482] In one embodiment of the application, the mixture can be cultured for a number of hours (about 3 hours) to about 14 days or any integral value of hours therebetween. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the application, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. It can also be desirable to have several cycles of stimulation, such that the culture time of the T cells can be 60 days or more. Conditions suitable for T cell culture include appropriate media (e.g., Minimal Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which can contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives for cell growth known to one of skill in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which is added with amino acids, sodium pyruvate, and vitamins, which is serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and / or an amount of cytokines sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures, not in cell cultures to be infused into a subject. The target cells are maintained under conditions necessary to support growth, e.g., at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0483] T cells exposed to different times of stimulation can exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product has a population of helper T cells (T H , CD4 + ) that is greater than the population of cytotoxic or suppressor T cells (T C , CD8 + ). Expansion of T cells ex vivo by stimulation of CD3 and CD28 receptors produces a population of T cells that is primarily composed of T H cells before about day 8-9, while after about day 8-9, the population of T cells contains an increasing population of T C cells. Thus, depending on the purpose of the treatment, it can be advantageous to infuse a subject with a population of T cells that contains primarily T H cells. Similarly, if an antigen-specific T CCell subpopulations, and it can be beneficial to expand the subpopulation to a greater extent.

[0484] In addition, other phenotypic markers change significantly in addition to CD4 and CD8 markers, but are largely reproducible during the cell expansion process. Thus, this reproducibility enables the ability to tailor activated T cell products for specific purposes.

[0485] Figure 25

[0486] The present application provides methods of treating a subject having or suspected of having an mRAS-associated cancer. The methods can be used to treat any cancer associated with RAS mutations, such as mutations at position G12, including hematological malignancies, solid tumors, primary or metastatic tumors.

[0487] Exemplary tumor and cancer types that can be treated by way of the present application include, but are not limited to, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine cancer, thyroid cancer, acute myeloid leukemia (AML), urothelial carcinoma, gastric adenocarcinoma, and cervical adenocarcinoma, head and neck squamous cell carcinoma (SCC), diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, chronic lymphocytic leukemia (CLL), lung SCC, small cell lung cancer (SCLC), renal papillary carcinoma, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0488] In certain embodiments, the present application provides methods of inducing an immune response against mRAS in a subject. For example, administration of a composition described herein can be used to induce a specific immune response, including a T cell-mediated immune response, against mRAS and cancer cells expressing mRAS. In certain instances, inducing an immune response against mRAS results in inhibition of tumor growth and tumor cell death.

[0489] In one embodiment, the method comprises contacting the subject with a composition of the present application. For example, in certain embodiments, the method comprises contacting the subject with a composition comprising an antigenic mRAS peptide described herein, a nucleic acid molecule encoding an mRAS peptide described herein, or a cell modified to comprise or express an mRAS peptide described herein. In certain embodiments, the method comprises contacting the subject with a composition polypeptide comprising a TCR described herein, a nucleic acid molecule encoding a polypeptide comprising a TCR described herein, a cell modified to express a TCR described herein.

[0490] In certain embodiments, a subject is identified as having an HLA type that is associated with an mRAS peptide bound by a TCR. For example, as described herein, in certain instances, a TCR binds a particular mRAS peptide in the context of a particular HLA molecule. Accordingly, in certain embodiments, the methods comprise identifying a subject as having a particular HLA molecule, and then administering to the subject a composition comprising or encoding a TCR described herein. For example, in one embodiment, the methods comprise identifying a subject as having an HLA-A*1 1 :01 molecule, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds an mRAS peptide comprising VVGACGVGK (SEQ ID NO: 5), VVVGACGVGK (SEQ ID NO: 6), VVGADGVGK (SEQ ID NO: 7), VVVGADGVGK (SEQ ID NO: 8), VVGARGVGK (SEQ ID NO: 9), VVVGARGVGK (SEQ ID NO: 10), VVGAVGVGK (SEQ ID NO: 1 1 ), or VVVGAVGVGK (SEQ ID NO: 12). In one embodiment, the methods comprise identifying a subject as having a particular HLA molecule, and then administering to the subject a composition comprising or encoding a particular mRAS peptide described herein. For example, in one embodiment, the methods comprise identifying a subject as having an HLA-A*1 1 :01 molecule, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises VVGACGVGK (SEQ ID NO: 5), VVVGACGVGK (SEQ ID NO: 6), VVGADGVGK (SEQ ID NO: 7), VVVGADGVGK (SEQ ID NO: 8), VVGARGVGK (SEQ ID NO: 9), VVVGARGVGK (SEQ ID NO: 10), VVGAVGVGK (SEQ ID NO: 1 1 ), or VVVGAVGVGK (SEQ ID NO: 12).

[0491] In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 molecule and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGACGVGK (SEQ ID NO: 5), VVVGACGVGK (SEQ ID NO: 6), VVGADGVGK (SEQ ID NO: 7), VVVGADGVGK (SEQ ID NO: 8), VVGARGVGK (SEQ ID NO: 9), VVVGARGVGK (SEQ ID NO: 10), VVGAVGVGK (SEQ ID NO: 11), or VVVGAVGVGK (SEQ ID NO: 12). In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 molecule and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises VVGACGVGK (SEQ ID NO: 5), VVVGACGVGK (SEQ ID NO: 6), VVGADGVGK (SEQ ID NO: 7), VVVGADGVGK (SEQ ID NO: 8), VVGARGVGK (SEQ ID NO: 9), VVVGARGVGK (SEQ ID NO: 10), VVGAVGVGK (SEQ ID NO: 11), or VVVGAVGVGK (SEQ ID NO: 12).

[0492] In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 molecule and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising KLVVVGACGV (SEQ ID NO: 1), KLVVVGADGV (SEQ ID NO: 2), KLVVVGARGV (SEQ ID NO: 3), or KLVVVGAVGV (SEQ ID NO: 4). In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 molecule and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises KLVVVGACGV (SEQ ID NO: 1), KLVVVGADGV (SEQ ID NO: 2), KLVVVGARGV (SEQ ID NO: 3), or KLVVVGAVGV (SEQ ID NO: 4).

[0493] In one embodiment, the method comprises identifying a subject as having an HLA-B*07:02 molecule and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to a mRAS peptide comprising GACGVGKSAL (SEQ ID NO: 13), GADGVGKSAL (SEQ ID NO: 14), GARGVGKSAL (SEQ ID NO: 15), or GAVGVGKSAL (SEQ ID NO: 16). In one embodiment, the method comprises identifying a subject as having an HLA-B*07:02 molecule and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing a mRAS peptide, wherein the mRAS peptide comprises GACGVGKSAL (SEQ ID NO: 13), GADGVGKSAL (SEQ ID NO: 14), GARGVGKSAL (SEQ ID NO: 15), or GAVGVGKSAL (SEQ ID NO: 16).

[0494] In one embodiment, the method comprises identifying a subject as having a particular RAS mutation. For example, in one embodiment, the subject is identified as having a particular mutation at G12 relative to wild-type RAS. For example, in one embodiment, the method comprises identifying a subject as having a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS.

[0495] In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12C RAS mutation and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to a mRAS peptide comprising KLVVVGA C GV (SEQ ID NO: 1). In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12C RAS mutation and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing a mRAS peptide, wherein the mRAS peptide comprises KLVVVGA C GV (SEQ ID NO: 1).

[0496] In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising KLVVVGA D GV (SEQ ID NO: 2). In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises KLVVVGA D GV (SEQ ID NO: 2).

[0497] In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12R RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising KLVVVGA R GV (SEQ ID NO: 3). In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12R RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises KLVVVGA R GV (SEQ ID NO: 3).

[0498] In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising KLVVVGA V GV (SEQ ID NO: 4). In one embodiment, the method comprises identifying a subject as having an HLA-A*02:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises KLVVVGA V GV (SEQ ID NO: 4).

[0499] In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12C RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGA C GVGK (SEQ ID NO: 5) or VVGA C In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12C RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises VVGA C GVGK (SEQ ID NO: 5) or VVGA C GVGK (SEQ ID NO: 6).

[0500] In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVGA D In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises VVGA D GVGK (SEQ ID NO: 7) or VVGA D GVGK (SEQ ID NO: 8).

[0501] In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12R RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGA R GVGK (SEQ ID NO: 9) or VVGA RmRAS peptide of GVGK (SEQ ID NO: 10). In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12R RAS mutation, and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing a mRAS peptide, wherein the mRAS peptide comprises VVGA R GVGK (SEQ ID NO: 9) or VVGA C GVGK (SEQ ID NO: 10).

[0502] In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to a peptide comprising VVGA V GVGK (SEQ ID NO: 11) or VVGA V mRAS peptide of GVGK (SEQ ID NO: 12). In one embodiment, the method comprises identifying a subject as having an HLA-A*03:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing a mRAS peptide, wherein the mRAS peptide comprises VVGA V GVGK (SEQ ID NO: 11) or VVGA C GVGK (SEQ ID NO: 12).

[0503] In one embodiment, the method comprises identifying a subject as having an HLA-A*11:01 allele and a G12C RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to a peptide comprising VVGA C GVGK (SEQ ID NO: 5) or VVGA C mRAS peptide of GVGK (SEQ ID NO: 6). In one embodiment, the method comprises identifying a subject as having an HLA-A*11:01 allele and a G12C RAS mutation, and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing a mRAS peptide, wherein the mRAS peptide comprises VVGA C GVGK (SEQ ID NO: 5) or VVGA C GVGK (SEQ ID NO: 6).

[0504] In one embodiment, the method comprises identifying a subject as having an HLA-A*11:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGA D GVGK (SEQ ID NO: 7) or VVGA D GVGK (SEQ ID NO: 8). In one embodiment, the method comprises identifying a subject as having an HLA-A*11:01 allele and a G12D RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding an mRAS peptide, or a cell expressing an mRAS peptide, wherein the mRAS peptide comprises VVGA D GVGK (SEQ ID NO: 7) or VVGA D GVGK (SEQ ID NO: 8).

[0505] In one embodiment, the method comprises identifying a subject as having an HLA-A*11:01 allele and a G12R RAS mutation, and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, wherein the TCR specifically binds to an mRAS peptide comprising VVGA R GVGK (SEQ ID NO: 9) or VVGA R GVGK (SEQ ID NO: 10). In one embodiment, the method comprises identifying a subject as havin...

Claims

1. A composition comprising a T-cell receptor (TCR), wherein the T-cell receptor specifically binds to a mutant RAS (mRAS) peptide in the context of an HLA-A*11:01 molecule, wherein, The HLA-A*11:01 molecule contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:5-12. Wherein, (b) the T cell receptor comprises: a T cell receptor α variable region 12-1CDR1 composed of the amino acid sequence of SEQ ID NO:31, a T cell receptor α variable region 12-1CDR2 composed of the amino acid sequence of SEQ ID NO:32, a T cell receptor α variable region 12-1CDR3 composed of the amino acid sequence of SEQ ID NO:33, a T cell receptor β variable region 28CDR1 composed of the amino acid sequence of SEQ ID NO:37, a T cell receptor β variable region 28CDR2 composed of the amino acid sequence of SEQ ID NO:38, and a T cell receptor β variable region 28CDR3 composed of the amino acid sequence of SEQ ID NO:39; (d) The T-cell receptor comprises: a T-cell receptor α variable region 17CDR1 composed of the amino acid sequence of SEQ ID NO:45, a T-cell receptor α variable region 17CDR2 composed of the amino acid sequence of SEQ ID NO:46, a T-cell receptor α variable region 17CDR3 composed of the amino acid sequence of SEQ ID NO:47, a T-cell receptor β variable region 11-2CDR1 composed of the amino acid sequence of SEQ ID NO:50, a T-cell receptor β variable region 11-2CDR2 composed of the amino acid sequence of SEQ ID NO:51, and a T-cell receptor β variable region 11-2CDR3 composed of the amino acid sequence of SEQ ID NO:52; or (f) The T-cell receptor comprises: a T-cell receptor α variable region 39CDR1 composed of the amino acid sequence of SEQ ID NO:17, a T-cell receptor α variable region 39CDR2 composed of the amino acid sequence of SEQ ID NO:18, a T-cell receptor α variable region 39CDR3 composed of the amino acid sequence of SEQ ID NO:19, a T-cell receptor β variable region 20-1CDR1 composed of the amino acid sequence of SEQ ID NO:23, a T-cell receptor β variable region 20-1CDR2 composed of the amino acid sequence of SEQ ID NO:24, and a T-cell receptor β variable region 20-1CDR3 composed of the amino acid sequence of SEQ ID NO:

25.

2. A composition comprising isolated nucleic acid molecules, said isolated nucleic acid molecules encoding a T-cell receptor (TCR), said T-cell receptor specifically binding to a mutant RAS (mRAS) peptide in the context of an HLA-A*11:01 molecule. in, The HLA-A*11:01 molecule contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:5-12. Wherein, (b) the T cell receptor comprises: a T cell receptor α variable region 12-1CDR1 composed of the amino acid sequence of SEQ ID NO:31, a T cell receptor α variable region 12-1CDR2 composed of the amino acid sequence of SEQ ID NO:32, a T cell receptor α variable region 12-1CDR3 composed of the amino acid sequence of SEQ ID NO:33, a T cell receptor β variable region 28CDR1 composed of the amino acid sequence of SEQ ID NO:37, a T cell receptor β variable region 28CDR2 composed of the amino acid sequence of SEQ ID NO:38, and a T cell receptor β variable region 28CDR3 composed of the amino acid sequence of SEQ ID NO:39; (d) The T-cell receptor comprises: a T-cell receptor α variable region 17CDR1 composed of the amino acid sequence of SEQ ID NO:45, a T-cell receptor α variable region 17CDR2 composed of the amino acid sequence of SEQ ID NO:46, a T-cell receptor α variable region 17CDR3 composed of the amino acid sequence of SEQ ID NO:47, a T-cell receptor β variable region 11-2CDR1 composed of the amino acid sequence of SEQ ID NO:50, a T-cell receptor β variable region 11-2CDR2 composed of the amino acid sequence of SEQ ID NO:51, and a T-cell receptor β variable region 11-2CDR3 composed of the amino acid sequence of SEQ ID NO:52; or (f) The T-cell receptor comprises: a T-cell receptor α variable region 39CDR1 composed of the amino acid sequence of SEQ ID NO:17, a T-cell receptor α variable region 39CDR2 composed of the amino acid sequence of SEQ ID NO:18, a T-cell receptor α variable region 39CDR3 composed of the amino acid sequence of SEQ ID NO:19, a T-cell receptor β variable region 20-1CDR1 composed of the amino acid sequence of SEQ ID NO:23, a T-cell receptor β variable region 20-1CDR2 composed of the amino acid sequence of SEQ ID NO:24, and a T-cell receptor β variable region 20-1CDR3 composed of the amino acid sequence of SEQ ID NO:

25.

3. The composition according to claim 1 or 2, wherein, The mutant RAS (mRAS) peptide, relative to wild-type RAS, contains a mutation at the position corresponding to G12.

4. The composition according to claim 3, wherein, The mutations in the mutant RAS (mRAS) peptide, relative to the wild-type RAS, correspond to mutations selected from the group consisting of G12V, G12R, G12C, and G12D.

5. The composition according to claim 4, wherein, The wild-type RAS contains the amino acid sequence of SEQ ID NO:177 or SEQ ID NO:

193.

6. A cell modified to express a T-cell receptor (TCR), said T-cell receptor specifically binding to a mutant RAS (mRAS) peptide in the context of an HLA-A*11:01 molecule, wherein, The HLA-A*11:01 molecule contains at least one amino acid sequence selected from the group consisting of SEQ ID NO:5-12. Wherein, (b) the T cell receptor comprises: a T cell receptor α variable region 12-1CDR1 composed of the amino acid sequence of SEQ ID NO:31, a T cell receptor α variable region 12-1CDR2 composed of the amino acid sequence of SEQ ID NO:32, a T cell receptor α variable region 12-1CDR3 composed of the amino acid sequence of SEQ ID NO:33, a T cell receptor β variable region 28CDR1 composed of the amino acid sequence of SEQ ID NO:37, a T cell receptor β variable region 28CDR2 composed of the amino acid sequence of SEQ ID NO:38, and a T cell receptor β variable region 28CDR3 composed of the amino acid sequence of SEQ ID NO:39; (d) The T-cell receptor comprises: a T-cell receptor α variable region 17CDR1 composed of the amino acid sequence of SEQ ID NO:45, a T-cell receptor α variable region 17CDR2 composed of the amino acid sequence of SEQ ID NO:46, a T-cell receptor α variable region 17CDR3 composed of the amino acid sequence of SEQ ID NO:47, a T-cell receptor β variable region 11-2CDR1 composed of the amino acid sequence of SEQ ID NO:50, a T-cell receptor β variable region 11-2CDR2 composed of the amino acid sequence of SEQ ID NO:51, and a T-cell receptor β variable region 11-2CDR3 composed of the amino acid sequence of SEQ ID NO:52; or (f) The T-cell receptor comprises: a T-cell receptor α variable region 39CDR1 composed of the amino acid sequence of SEQ ID NO:17, a T-cell receptor α variable region 39CDR2 composed of the amino acid sequence of SEQ ID NO:18, a T-cell receptor α variable region 39CDR3 composed of the amino acid sequence of SEQ ID NO:19, a T-cell receptor β variable region 20-1CDR1 composed of the amino acid sequence of SEQ ID NO:23, a T-cell receptor β variable region 20-1CDR2 composed of the amino acid sequence of SEQ ID NO:24, and a T-cell receptor β variable region 20-1CDR3 composed of the amino acid sequence of SEQ ID NO:

25.

7. The cell according to claim 6, wherein, The mutant RAS (mRAS) peptide, relative to wild-type RAS, contains a mutation at the position corresponding to G12.

8. The cell according to claim 7, wherein, The mutations in the mutant RAS (mRAS) peptide, relative to the wild-type RAS, correspond to mutations selected from the group consisting of G12V, G12R, G12C, and G12D.

9. The cell according to claim 8, wherein, The wild-type RAS contains the amino acid sequence of SEQ ID NO:177 or SEQ ID NO:

193.

10. The cell according to any one of claims 6-9, wherein, The cells were modified to express a fusion polypeptide containing TCRα and TCRβ chains; The cells are genetically modified by introducing isolated nucleic acid molecules encoding polypeptides, wherein the polypeptides contain at least one of TCRα chains and TCRβ chains; The cells in question are immune cells; Wherein, the cells are autologous to the subject suffering from RAS-related cancer; or The cells in question are autologous to the subjects who have HLA-A*11:

01.

11. The cell according to claim 10, wherein, The immune cells are selected from the group consisting of T cells, NK cells, and NK T cells.

12. Use of the composition of any one of claims 1-5 or the cell of any one of claims 6-11 in the preparation of a medicament for treating a subject with mRAS-related cancer or for inducing an immune response in a subject with mRAS-related cancer.

13. The use according to claim 12, wherein, The treatment or the induction includes identifying the subject's HLA type.

14. The use according to claim 12 or 13, wherein, The treatment or the induction includes: isolating one or more cells of the subject and modifying the one or more cells to express TCR.

15. The use according to claim 12 or 13, wherein, The treatment or the induction comprises: modifying one or more cells to express TCR by contacting one or more cells with isolated nucleic acid molecules, the isolated nucleic acid molecules encoding one or more of a TCRα chain and a TCRβ chain.

16. The use according to claim 12 or 13, wherein, The cancers associated with mRAS are selected from the group consisting of: pancreatic cancer, pancreatic ductal adenocarcinoma, colon cancer, colorectal adenocarcinoma, myeloma, multiple myeloma, lung adenocarcinoma, melanoma, uterine cancer, thyroid cancer, acute myeloid leukemia, urothelial carcinoma, gastric adenocarcinoma and cervical adenocarcinoma, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal adenocarcinoma, chronic lymphocytic leukemia, lung squamous cell carcinoma, small cell lung cancer, renal papillary carcinoma, hepatocellular carcinoma, breast cancer, cervical squamous cell carcinoma, ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme, medulloblastoma, renal cell carcinoma, esophageal squamous cell carcinoma, osteosarcoma, sarcoma and small intestinal neuroendocrine tumors.

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