Compositions and methods for targeting mutant ras

Abstract

To provide compositions and methods of treating cancer associated with mutant RAS.SOLUTION: The invention relates to compositions and methods of treating cancer associated with mutant RAS. In certain aspects, the 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.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 796,733, filed January 25, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] Somatic mutations have been identified as common drivers of carcinogenesis. 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 cancers and are involved in the pathogenesis of a variety of highly prevalent malignancies, including lung, colorectal, and pancreatic ductal adenocarcinoma. Mutant RAS is an attractive target for cancer therapy because it is uniquely expressed by cancer cells and is considered a critical driver mutation for tumor growth and survival. These mutations usually involve the codon 12 position of the RAS protein, and the amino acid changes are highly conserved and most often result from amino acid substitutions of G12C, G12D, G12R, and G12V. Pathological RAS mutations are gain-of-function mutations that cause constitutive activation of intracellular GTPase signaling that promotes cell proliferation. RAS mutations may be found at high frequency in certain types of cancer. For example, G12D and G12V mutations are present in 60% to 70% of pancreatic cancers and 20% to 30% of colorectal cancers.Unfortunately, there are no effective pharmacological inhibitors of the RAS oncoproteins.

[0003] Thus, there is a need in the art for compositions and methods for treating mutant RAS-associated cancers. The present invention addresses and meets these and other needs. Summary of the Invention

[0004] In one aspect, the invention provides an immunogenic composition comprising a mutant RAS peptide comprising a mutation 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.

[0005] In one embodiment, the mutant RAS peptide is selected from the group consisting of SEQ ID NO:1 to 1 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 SEQ ID NO:1 to SEQ ID NO:6. 1 6.

[0006] In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a mutant RAS peptide comprising a mutation relative to G12 of wild-type RAS.

[0007] In one embodiment, the invention provides a cell that comprises or is engineered to express a mutant RAS peptide that comprises a mutation 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, and a NK T cell.

[0008] In one aspect, the present invention provides a method for inducing an immune response in a subject comprising administering to the subject an immunological composition comprising a mutant RAS peptide comprising a mutation at a relative position to G12 of wild-type RAS, or a nucleic acid molecule encoding a mutant RAS peptide comprising a mutation at a relative position to G12 of wild-type RAS.

[0009] In one embodiment, the method comprises identifying the HLA type of a subject, and administering to the subject a composition comprising or encoding a mutant RAS peptide comprising a mutation at a relative position 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal gland cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (ESCC), and / or esophageal squamous cell carcinoma (ESC). SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0010] In one aspect, the present invention provides a method for inducing an immune response in a subject, comprising contacting a cell with a composition comprising a mutant RAS peptide comprising a mutation at a relative position to G12 of wild-type RAS, thereby stimulating the cell; and administering the stimulated cell to the subject.In one embodiment, the method comprises contacting a naive T cell of a subject with an antigen-presenting cell that presents a mutant RAS peptide, thereby stimulating the T cell.In one embodiment, the cell is autologous to the subject.In one embodiment, the T cell and the antigen-presenting cell are autologous to the subject.

[0011] In one aspect, the present invention provides a composition comprising 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 RAS peptide comprises a mutation at a position corresponding to G12 compared to wild-type RAS. In one embodiment, the mutation in the mRAS peptide corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V compared to wild-type RAS.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In one embodiment, the composition comprises a fusion polypeptide comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the fusion polypeptide comprises a linker domain. In one embodiment, the linker domain is a cleavable linker domain.

[0019] In one embodiment, the present invention 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.

[0020] In one aspect, the present invention 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 compared to wild-type RAS. In one embodiment, the mutation in the mRAS peptide corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V compared to wild-type RAS.

[0021] In one embodiment, the cell is modified to express a fusion polypeptide comprising a TCR alpha chain and a TCR beta 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 alpha chain and a TCR beta 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, a NK cell, and a NK T cell. In one embodiment, the cell is autologous to the subject having a cancer associated with RAS. In one embodiment, the cell is autologous to the 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.

[0022] In one embodiment, the invention provides a method of treating a subject having a cancer associated with mRAS, comprising administering to the subject cells 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 is diagnosed with 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal gland cancer, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (ESC), esophageal squamous cell carcinoma (ESC), esophageal adenocar ... The patient has a cancer selected from the group consisting of SCC, osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

[0023] In one embodiment, the method includes identifying the HLA type of the subject. In one embodiment, the method includes isolating one or more cells of the subject and modifying the one or more cells to express a TCR. In one embodiment, the method includes 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 the TCR alpha and beta chains.

[0024] The foregoing summary, as well as the following detailed description of exemplary embodiments of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows a schematic diagram illustrating the discovery strategy for mutated RAS (mRAS) epitopes. [Diagram 2] FIG. 1 shows a schematic diagram of an exemplary computational method used to predict neoepitopes of mRAS. [Diagram 3] 1 shows the results of an exemplary experiment demonstrating the predicted affinity of mRAS peptides for various HLA molecules. [Figure 4A] We present the results of an exemplary experiment showing the predicted affinity of the G12m RAS peptide for various HLA molecules. Figure 4A shows a heatmap representing computational predictions of mRAS epitopes with affinity below 500 nM using antigen.garnish. We also present the HLA frequency in the US population, and the KRAS mutation frequency occurring in pancreatic adenocarcinoma (PDA), colorectal cancer (CRC), and lung adenocarcinoma (LAC). [Figure 4B] 4A-4C show the results of an exemplary experiment demonstrating the predicted affinity of the G12m RAS peptide for various HLA molecules. FIG. 4B shows a table summarizing the predicted binding of mRAS epitopes to various HLA molecules. [Diagram 5]1 shows the results of an exemplary experiment using a fluorescence polarization assay to determine peptide-MHC binding, demonstrating the affinity of various mRAS peptides for specific HLA molecules. [Figure 6] Figures 6A and 6B show the results of an exemplary experiment providing a biochemical assessment of mRAS epitope binding. (Figure 6A) Competitive peptide binding fluorescence polarization assay. Strong binding affinity is shown with log[IC50]<3.7 (dashed line). (Figure 6B) Peptide stability by scintillation proximity assay. The stability of the published T cell epitope is shown by the grey area (range) and dashed line (mean). [Figure 7] Figures 7A-E show the results of experiments using the generated monoallelic RAS tandem minigene (TMG) cell lines. (Figure 7A) Schematic of the lentiviral vector construct. (Figure 7B) FACS plot of HLA / RAS TMG modified K56 cell lines shown by mCherry and GFP positivity. Validation of (Figure 7C) HLA class I and (Figure 7D) HLA specific expression of RAS TMG cell lines by FACS. Figure 7E shows a table of wild type and mRAS long peptide sequences, as well as viral control peptides encoded by the RAS TMG constructs. [Figure 8]8A-8J show the results of an example experiment demonstrating HLA class I immunoprecipitated peptide elution and detection of mRAS epitopes by tandem mass spectrometry (MS / MS). (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 8I) A*11:01-restricted RAS G12R epitope VV_R. (Figure 8J) B*07:02-restricted RAS G12R epitope GA_R. [Figure 9] FIG. 1 is a schematic diagram outlining mRAS epitopes detected by mass spectrometry, where the shaded boxes represent the binding between the epitopes and HLA molecules. [Figure 10] Results of an exemplary experiment comparing predicted and detected mRAS epitopes in the context of specific HLA types are shown. Peptides highlighted in red are computationally predicted epitopes detected using p / MHC IP HPLC tandem mass spectrometry. [Figure 11] An experimental schematic showing protocol details for generating and identifying mRAS-specific CD8+ T cells is shown. [Figure 12] 1 shows exemplary experimental results outlining mRAS-specific CD8+ T cell responses in healthy donors. [Figure 13]Figures 13A-F show the results of an exemplary experiment demonstrating the antigenicity of mRAS epitopes: (Figure 13A) IFN-γ ELISPOT of A*03:01-restricted mRAS epitope responses, (Figure 13B) A*11:01-restricted mRAS epitope responses, and (Figure 13C) B*07:02-restricted mRAS epitope responses. (Figures 13D-F) Representative peptide-MHC multimer staining results for donors highlighted with red symbols. [Figure 14] 1 shows the results of an exemplary experiment demonstrating detection of mRAS-specific CD8+ by peptide / MHC multimer staining detection. [Figure 15] 1 shows the results of an exemplary experiment demonstrating that mRAS T cell responses are highly specific for the mRAS peptide of interest and show no cross-reactivity to wild-type RAS peptide. [Figure 16] We present the results of an exemplary experiment demonstrating that the B7-G12R response is high affinity, as demonstrated by IFN-γ secretion and cytotoxicity assays. Importantly, no reactivity was detected against cell lines expressing wild-type or alternatively mutated RAS peptides. [Figure 17] 1 shows the results of an exemplary experiment demonstrating that HLA-B*07:02-restricted RAS G12R-specific CD8+ T cells, when genetically modified to express HLA-B*07:02, exhibit cytotoxicity against PSN, a PDA cell line with endogenous RAS G12R expression. [Figure 18] 1 shows the results of an exemplary experiment demonstrating the identification of mRNA-specific TCR sequences. [Figure 19] 1 shows the design of lentiviral constructs for TCR831 and TCR833. [Figure 20] A table summarizing additional TCR constructs, their KRAS mutation specificity, HLA restriction, alpha and beta chain identity, and associated CDR3 amino acid sequences is provided. [Figure 21] 1 shows the results of an exemplary experiment demonstrating transgenic expression of TCR831 and TCR833 in primary CD8+ cells. [Figure 22]The results of an exemplary experiment are shown demonstrating that transgenic TCR831 and TCR833 have high affinity for HLA-A*11:01 restricted KRAS G12V and are unresponsive to wild-type RAS antigens. Furthermore, TCR831 and TCR833 recognize antigens endogenously processed and presented by K562-A*11:01 cells genetically modified to express a RASmg construct. [Figure 23] 1 shows the results of an exemplary experiment demonstrating that transgenic expression of TCR831 and TCR833 confers cytotoxicity to K562-A*11:01 cells expressing the RAS G12V peptide of the endogenous RASmg construct but not the wild-type RAS G12V peptide. [Figure 24]

[0036] Figure 1 shows the results of 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. [Diagram 25]Figures 25A-G show the results of experiments characterizing TCR831 expression and function. (Figure 25A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 25B) Evaluation of TCR binding activity by Jurkat reporter cells. (Figure 25C) Evaluation of TCR specificity and cross-reactivity to alternative mutant KRAS epitopes by Jurkat reporter assay. TCR831 shows specificity for RAS G12V (VVV_V) but not wild type. Cross-reactivity was observed for RAS G12C (VVV_C). (Figure 25D) TCR activation of Jurkat reporter cells after co-culture with A*11:01 positive RAS G12V tumor cell line. (Figure 25E) Expression of TCR831 in primary CD8+ T cells. (Figure 25F) 4 hour 51Cr assay results show 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 25G) 4 hour 51Cr assay results show specific lysis of A*11:01 positive RAS G12V tumor cell line with effector to target ratio of 10:1. Cell line colors correspond to those in Figure 25C. [Figure 26]Figures 26A-G show the results of example experiments characterizing TCR833 expression and function. (Figure 26A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 26B) Assessment of TCR binding activity by Jurkat reporter cells. (Figure 26C) Assessment of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR831 shows specificity for RAS G12V (VVV_V) but not wild type. Cross-reactivity was observed for RAS G12C (VVV_C). (Figure 26D) TCR activation of Jurkat reporter cells after co-culture with A*11:01 positive RAS G12V tumor cell line. (Figure 26E) Expression of TCR833 in primary CD8+ T cells. (FIG. 26F) 4-hour 51Cr assay results showing specific lysis of K562-A*11:01 cells pulsed with G12V peptide (blue) and expressing RAS TMG constructs (red) but not wild type (black). (FIG. 26G) 4-hour 51Cr assay results showing specific lysis of A*11:01 positive RAS G12V tumor cell line. [Figure 27] Figures 27A-C show the results of example experiments characterizing TCR897 expression and function. (Figure 27A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 27B) Assessment of TCR binding activity by Jurkat reporter cells. (Figure 27C) Assessment of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR897 shows specificity for RAS G12V (VV_V), but not wild type. Cross-reactivity to RAS G12C (VV_C) and G12D (VV_D) epitopes was observed. [Figure 28]Figures 28A-G show the results of example experiments characterizing TCR896 expression and function. (Figure 28A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 28B) Assessment of TCR binding activity by Jurkat reporter cells. (Figure 28C) Assessment of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR896 shows specificity for RAS G12V (VVV_V), but not wild type. (Figure 28D) TCR activation of Jurkat reporter cells after co-culture with A*03:01 positive RAS G12V tumor cell line. (Figure 28E) Expression of TCR896 in primary CD8+ T cells. (FIG. 28F) 4-hour 51Cr assay results showing specific lysis of K562-A*03:01 cells pulsed with G12V peptide (blue) or expressing RAS TMG constructs (red) but not wild type (black). (FIG. 28G) 4-hour 51Cr assay results showing specific lysis of A*03:01 positive RAS G12V tumor cell lines. [Figure 29] Figures 29A and 29B show the results of example experiments characterizing TCR847 expression and function. (Figure 29A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 29B) Assessment of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR847 shows specificity for RAS G12R (GA_R) but not for wild-type or alternative mutant RAS epitopes. [Diagram 30]Figures 30A-E show the results of example experiments characterizing TCR864 expression and function. (Figure 30A) Verification of TCR expression by peptide-MHC multimer staining of lentivirally transduced Jurkat reporter cells. (Figure 30B) Assessment of TCR binding activity by Jurkat reporter cells. (Figure 30C) Assessment of TCR specificity and cross-reactivity to alternative mutant RAS epitopes by Jurkat reporter assay. TCR864 shows specificity for RAS G12R (GA_R) but not wild type or alternative mutant RAS epitopes. (Figure 30D) Expression of TCR864 in primary CD8+ T cells. (Figure 30E) Results of a 4-hour 51Cr assay show specific lysis of K562-B*07:02 cells pulsed with G12R peptide (blue) or expressing the RAS TMG construct (red) but not wild type (black). [Diagram 31] FIG. 1 shows a schematic diagram of a clinical trial using dendritic cell (DC) vaccination against a short mRAS peptide. [Diagram 32] FIG. 1 shows a schematic of the experimental process used to identify the mRAS TCR in vaccinated PDA patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention The present invention relates to compositions and methods for treating cancers associated with mutated RAS (mRAS). Somatic mutations within RAS provide a form of non-self antigen, making RAS-mutated tumors amenable to immune-based therapeutic approaches, including but not limited to adoptive T cell therapy. T cells have unique T cell receptors (TCRs) that can recognize subtle mutations within intracellular proteins that may be expressed and presented on HLA molecules by tumor cells.

[0027] The present invention is applicable to any member of the RAS family of oncogenic proteins, including but not limited to KRAS, NRAS, and HRAS. The RAS hotspot mutations described herein (e.g., mutations at position G12) are common among KRAS, NRAS, and HRAS-related cancers. Furthermore, the amino acid sequences of the RAS peptides described herein are conserved among all RAS family members. Thus, the mutant RAS peptides and TCRs described herein are applicable to induce immune responses against mutant RAS family members to treat cancers associated with mutant RAS family members. As used herein, "RAS" is meant to include any member of the RAS family of proteins.

[0028] The present invention 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 for inducing immune responses against mRAS.In certain embodiments, the present invention relates to immunogenic compositions, such as vaccines, that comprise the antigenic mRAS peptides described herein or the nucleic acid molecules that code for the antigenic mRAS peptides described herein.

[0029] The present invention 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 the context of prevalent HLA types. In certain aspects, the present invention relates to compositions comprising isolated TCRs or nucleic acid molecules encoding isolated TCRs, wherein the isolated TCRs specifically bind to RAS, mRAS, or fragments thereof. In one embodiment, the compositions comprise cells, e.g., autologous or allogeneic T cells, genetically modified to express a TCR that specifically binds to RAS, mRAS, or fragments thereof.

[0030] In a particular aspect, the present invention relates to a method for treating or preventing mRAS-associated cancer using antigenic mRAS peptides or TCRs as 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 as 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, loaded with one or more mRAS peptides or one or more nucleic acid molecules encoding one or more mRAS peptides as described herein. In a particular embodiment, the present invention relates to a method using TCR therapy, e.g., adoptive TCR therapy. In one embodiment, the method comprises administering to a subject with an mRAS-associated cancer at least one T cell genetically modified to express a TCR that specifically binds to RAS, mRAS, or a fragment thereof.

[0031] Exemplary mRAS-associated cancers treatable by the compositions and methods of the invention include 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (ESCC), and esophageal squamous cell carcinoma (ESC). SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumors (NETs).

[0032] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0033] As used herein, each of the following terms has the meaning associated with it in this section.

[0034] The articles "a" and "an" are used herein to refer to one or to 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.

[0035] As used herein, the terms "inhibit" and "inhibiting" refer to reducing, suppressing, decreasing, or blocking an activity or function by at least about 10% compared 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%.

[0036] The terms "effective amount" and "pharmacologically effective amount" refer to an amount of an agent sufficient to provide a desired biological result. The result may be the reduction and / or alleviation of the signs, symptoms, or pathology of a disease or disorder, or 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.

[0037] 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 humans in need of treatment or susceptible to a condition or its sequelae. Individuals may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, as well as mice and humans.

[0038] The term "abnormal" when used in reference to organisms, tissues, cells, or components thereof, refers to 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 exhibit the "normal" (expected / homeostatic) respective characteristic. A characteristic that is normal or expected in one cell, tissue type, or subject may be abnormal in another cell or tissue type.

[0039] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, among other things, to T cells undergoing cell division.

[0040] A "disease" is a state of a subject's health in which the subject is unable to maintain homeostasis and if the disease is not ameliorated, the subject's health continues to deteriorate.

[0041] In contrast, a "disorder" in a subject is a health state in which the subject is able to maintain homeostasis, but in which the subject's health state is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause the subject's health state to be further impaired.

[0042] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such sign or symptom is experienced by a patient, or both, are reduced.

[0043] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal 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, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0044] The term "anti-tumor effect" as used herein refers to a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life span, or an improvement in various physiological symptoms associated with a cancerous condition. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent the development of tumors in the first place.

[0045] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into that individual.

[0046] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0047] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0048] An "effective amount" or "therapeutically effective amount" of a compound is the amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0049] As used herein, "instructional material" includes publications, records, diagrams, or any other means of expression that can be used to communicate the utility of the compounds, compositions, vectors, or delivery systems of the invention in the kit to provide relief from various diseases or disorders described herein. Optionally, or alternatively, the instructional material can describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructional material of the kit of the invention can be, for example, affixed to a container containing the identified compounds, compositions, vectors, or delivery systems of the invention or shipped together with a container containing the identified compounds, compositions, vectors, or delivery systems. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compounds are used cooperatively by the recipient.

[0050] As used herein, "operably linked" or "operatively linked" may mean that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as 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, modifications of this distance can be accommodated without loss of promoter function.

[0051] A "therapeutic treatment" is a treatment administered to a subject who exhibits symptoms of a disease or disorder with the intent of reducing or eliminating those symptoms.

[0052] 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.

[0053] As used herein, the phrases "biological sample," "sample," or "specimen" are intended to include any sample containing cells, tissues, or bodily fluids in which expression of a nucleic acid or polypeptide can be detected. A biological sample may include any biological material suitable for detecting a desired biomarker, and may include cellular and / or non-cellular 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 "body fluids." Biological samples can be obtained from a patient by a variety of techniques, such as, for example, scraping or swabbing an area or using a needle to obtain a body fluid. Methods for collecting various body samples are well known in the art.

[0054] "CDR" is defined as the complementarity determining region of the amino acid sequence of a TCR or a TCR chain.

[0055] As used herein, "immunoassay" refers to any binding assay that uses an antibody that can specifically bind to a target molecule to detect and quantitate the target molecule.

[0056] The term "specifically binds" as used herein with respect to a polypeptide (e.g., a TCR or a TCR chain) refers to a polypeptide that recognizes and binds to a particular target molecule but does not substantially recognize or bind other molecules in a sample. In some cases, the terms "specific binding" or "specifically binding" are used to mean that recognition and binding is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule.

[0057] The "coding region" of a gene consists of nucleotide residues on the coding strand of the gene and the nucleotides on the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of the mRNA molecule generated by transcription of the gene.

[0058] A "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that coincide with the anticodon region of a transfer RNA molecule during translation of the mRNA molecule, or that code for a stop codon. Thus, a coding region may 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 translocation signal sequence).

[0059] "Differentially decreased expression" or "downregulation" refers to a biomarker product level that 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, than a control.

[0060] "Differentially increased expression" or "upregulation" refers to a biomarker product level that 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, over a control.

[0061] "Complementary" as used herein to refer to nucleic acids refers to the 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 can form specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region that is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand can base pair with a residue of a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid such that at least one nucleotide residue of the first region can base pair with a residue of the second region when the two regions are arranged in an antiparallel manner. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, such that when the first and second portions are positioned antiparallel, 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 can base pair with nucleotide residues of the second portion. In some embodiments, all nucleotide residues of the first portion can base pair with nucleotide residues of the second portion.

[0062] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0063] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0064] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase protein-encoding nucleotide sequence or RNA may also contain introns to the extent that a protein-encoding nucleotide sequence may contain introns in some versions.

[0065] The term "hybridoma" as used herein refers to a cell resulting from the fusion of a B lymphocyte with a fusion partner, such as a myeloma cell. Hybridomas can be cloned and maintained indefinitely in cell culture and can produce monoclonal antibodies. Hybridomas can also be considered as hybrid cells.

[0066] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in its normal context in a living subject is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0067] "Isolated nucleic acid" refers to a nucleic acid segment or fragment separated from sequences adjacent to it in its naturally occurring state, i.e., a DNA fragment removed from sequences that normally flank the fragment, i.e., sequences adjacent to the fragment in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., the RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (i.e., as a cDNA, or as a genome or cDNA generated by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0068] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0069] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotide as used herein 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 recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and by synthetic means.

[0070] "Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. They are able to deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of 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.

[0071] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed 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 may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0072] The term "RNA" as used herein is defined as ribonucleic acid.

[0073] The term "recombinant DNA" as used herein is defined as DNA that is produced by joining fragments of DNA from different sources.

[0074] The term "recombinant polypeptide," as used herein, is defined as a polypeptide produced using recombinant DNA methods.

[0075] As used herein, "linked" refers to the covalent attachment of one molecule to a second molecule.

[0076] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of 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 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, the comparison is performed when the two sequences are aligned to give maximum homology.

[0077] A "variant" as the term is used herein is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains essential biological properties of the reference molecule. The sequence changes of the nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. The sequence changes of the peptide variant are usually limited or conservative, so that the sequences of the reference peptide and the variant are generally very similar and identical in many regions. The variant and the reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The nucleic acid or peptide variant may be a naturally occurring variant, such as an allelic polymorphism, or may be a variant that is not known to occur in nature. Non-naturally occurring nucleic acid and peptide variants can be generated by mutagenesis techniques or direct synthesis. In various embodiments, the 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 the reference sequence.

[0078] The term "modulate" as used herein may refer to any manner of altering the level or activity of a substrate. Non-limiting examples of modulation for proteins include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting protein localization. Non-limiting examples of modulation for enzymes further include affecting enzyme activity. "Modulator" refers to a molecule whose activity includes affecting the level or activity of a substrate. Modulators may be direct or indirect. Modulators may function to activate, inhibit, or otherwise regulate their substrate.

[0079] As used herein, a "scanning window" refers to a segment of several adjacent positions within which a sequence may be evaluated independently of any adjacent sequences. The scanning window is typically moved incrementally along the length of the sequence being evaluated, with each new segment being evaluated individually. The incremental shift may be to one or more positions.

[0080] As used herein, "vector" may refer to a nucleic acid sequence that includes an origin of replication. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector that is integrated into a host genome.

[0081] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types with which it is normally associated in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that are 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.

[0082] Ranges: Throughout this disclosure, various aspects of the invention 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 invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0083] explanation The present invention relates to compositions and methods for treating mRAS-associated 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 severity, and the like.

[0084] In one aspect, the invention relates to an immunogenic composition 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 comprising a mutation relative to G12 of 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 compared to wild-type RAS.

[0085] In one embodiment, the invention provides an isolated nucleic acid molecule encoding an mRAS peptide described herein. In one embodiment, the invention provides a cell, e.g., an antigen-presenting cell, that comprises an mRAS peptide described herein or a nucleic acid molecule encoding an mRAS peptide.

[0086] In one aspect, the invention relates to a composition comprising a polypeptide comprising one or more TCR chains (e.g., TCR alpha, TCR beta, TCR delta, and TCR gamma) that, alone or together, specifically binds to RAS, mRAS, or a fragment thereof. In one embodiment, the composition comprises a TCR comprising a TCR alpha and a TCR beta chain, wherein the TCR specifically binds to RAS, mRAS, or a fragment thereof. Hereinafter, reference to "TCR" refers to the heterodimeric T cell receptor, the individual T cell receptor chains (e.g., TCR alpha, TCR beta, TCR delta, and TCR gamma chains), and functional portions and variants thereof.

[0087] In one embodiment, the TCR specifically binds to mRAS that includes a mutation at a position relative to G12 of wild-type RAS. For example, in a particular embodiment, the TCR specifically binds to mRAS that includes a G12C, G12D, G12R, or G12V mutation compared to wild-type RAS. In a particular embodiment, the TCR specifically binds to a fragment of mRAS, where the fragment includes a mutation at a position corresponding to G12. In a particular embodiment, the TCR specifically binds to an mRAS fragment in the context of a particular HLA type. In one embodiment, the composition comprises a fusion polypeptide that includes a TCR alpha chain and a TCR beta chain, where the TCR alpha chain and the TCR beta chain together form a heterodimeric TCR. In one embodiment, the fusion polypeptide comprises a cleavable linker between the TCR alpha chain and the TCR beta chain.

[0088] In one aspect, the invention provides an isolated nucleic acid molecule encoding a TCR as described herein.In one aspect, the invention provides a cell, such as a T cell, that has been modified to express a TCR as described herein.

[0089] In one embodiment, the present invention provides a method for treating or preventing mRAS-associated cancer in a subject having, suspected of having, or at risk of having mRAS-associated cancer. Exemplary mRAS-associated cancers treatable or preventable by the compositions and methods of the present invention include 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical squamous cell carcinoma (CLL), and uterine cancer. SCC), ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (esophageal SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumors (NETs).

[0090] In one embodiment, the method includes administering to the subject an immunogenic composition comprising at least one cell comprising an mRAS peptide, a nucleic acid molecule encoding an mRAS peptide, or a nucleic acid molecule encoding an mRAS peptide. In one embodiment, the method includes administering antigen-presenting cells, such as dendritic cells, 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 cells are autologous cells or are derived from autologous cells. For example, in one embodiment, the method includes isolating autologous cells from the subject; culturing the autologous cells ex vivo; loading the isolated autologous cells with one or more mRAS peptides or a nucleic acid molecule encoding one or more mRAS peptides, thereby generating antigen-presenting cells that present the mRAS peptides described herein; and administering the antigen-presenting cells to the subject. In certain embodiments, the particular type of mRAS peptide used in the method depends on the particular HLA type of the subject or cell.

[0091] 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 both adoptive TCRs, wherein autologous T cells are genetically modified to express a TCR as 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 as described herein; and administering the genetically modified cells to the subject. In certain embodiments, the particular type of TCR used in the method depends on the particular HLA type of the subject or cells.

[0092] mRAS Peptides and Vaccines In some embodiments, the invention provides compositions comprising an antigenic mRAS peptide. In one embodiment, the mRAS peptide stimulates or induces an anti-mRAS immune response in a subject.

[0093] In one embodiment, the mRAS peptide comprises a mutation relative to G12 of wild-type RAS, hi one embodiment, the mRAS peptide comprises a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS.

[0094] In one embodiment, the mRAS peptide has 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 invention, the mRAS peptide comprises a mutation relative to G12 of wild-type RAS, wherein the mRAS peptide has 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.

[0095] Exemplary antigenic mRAS peptides of the invention are provided in Table 1.

[0096] [Table 1]

[0097] In one embodiment, the present invention provides an immunogenic composition 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, it must induce an immune response against mRAS in a cell, tissue, or mammal (e.g., human). In certain examples, the vaccine induces a protective immune response in a mammal. As used herein, an "immunogenic composition" may include an antigen (e.g., an mRAS peptide), a nucleic acid encoding the antigen, a cell expressing or presenting the antigen or a cellular component, or a combination thereof. In certain embodiments, the composition includes or encodes all or a portion of any peptide antigen described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is a mixture that includes an additional immunostimulatory agent or a nucleic acid encoding such an agent. The immunostimulatory agent includes, but is not limited to, an additional antigen, an immunomodulatory agent, an antigen-presenting cell, a lipid nanoparticle, or an adjuvant. In other embodiments, one or more additional agents are covalently attached to the antigen or the immunostimulatory agent, in any combination.

[0098] In the context of the present invention, the term "vaccine" refers to a composition that induces an immune response upon inoculation into an animal. In some embodiments, the induced immune response provides protective immunity.

[0099] Vaccines of the present invention may vary in composition in their nucleic acid and / or cellular components. In a non-limiting example, vaccines that contain or code for mRAS peptide antigens can also be formulated with adjuvants. Of course, it will be understood that the various compositions described herein may further contain additional components. For example, one or more vaccine components may be contained in lipids, liposomes, or lipid nanoparticles. In another non-limiting example, vaccines may contain one or more adjuvants. Exemplary adjuvants include, but are not limited to, alpha interferon, gamma interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, 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 may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, 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, and fibroblast growth factor. child, 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, KILLE R, 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, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and their functional fragments.

[0100] The vaccine of the present invention and its various components may be prepared and / or administered by any of the methods disclosed herein or as would be known to one of skill in the art in light of the present disclosure.

[0101] The induction of immunity by an mRAS peptide antigen can be detected by observing the response of all or part of the host's immune system against mRAS in vivo or in vitro.

[0102] The present invention includes cells that have been exposed to 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 Ag-loaded in vitro, e.g., by culturing ex vivo in the presence of antigen, or in vivo by exposure to antigen.

[0103] Those skilled in the art will also readily understand that APCs can be "pulsed" in a manner that exposes the APC to an antigen for a sufficient time to promote the presentation of that antigen on the surface of the APC. For example, APCs can be exposed to an antigen in the form of a small peptide fragment called an antigen peptide. This is "pulsed" directly on the outside of the APC. Alternatively, APCs can be incubated with the antigen peptide and ingested. The APC then presents the antigen peptide on the surface of the APC. Antigens in peptide form can be exposed to cells by standard "pulsing" techniques described herein and known in the art.

[0104] The antigen-loaded APC, also known as the "pulsed APC" of the present invention, is generated by exposing the APC to antigen either in vitro or in vivo. When the APC is pulsed in vitro, the APC can be plated in a culture dish and exposed to antigen in a sufficient amount and for a sufficient time to allow the antigen to bind to the APC. The amount and time required to achieve antigen binding to the APC can be determined using methods known in the art or disclosed herein. Other methods known to those skilled in the art, such as immunoassays or binding assays, can be used to detect the presence of antigen on the APC after exposure to antigen.

[0105] In a further embodiment of the present invention, APC can be transfected with vector that allows APC to express specific peptide.The peptide expressed by APC can then be processed and presented on cell surface on MHC receptor.The transfected APC can then be used as an immunogenic composition to generate immune response against the protein encoded by vector.

[0106] As discussed elsewhere herein, vectors can be prepared containing specific polynucleotides that encode and express peptides against which an 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.

[0107] In another embodiment, the viral vector can be targeted to the APC by modifying it to encode a protein or portion thereof that is recognized by a receptor on the APC, such that occupation of the APC receptor by the vector initiates endocytosis of the vector, allowing processing and presentation of the antigen encoded by the viral vector's nucleic acid.

[0108] As contemplated herein, various methods can be used to transfect a polynucleotide into a host cell. Methods include, but are not limited to, calcium phosphate precipitation, lipofection, particle guns, microinjection, electroporation, colloidal dispersion systems (i.e., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes). These methods are understood in the art and are described in the published literature to enable those skilled in the art to carry out these methods.

[0109] In another embodiment, the polynucleotide encoding the antigen can be cloned into an expression vector, and the vector can be introduced into APC to generate loaded APC.Various types of vectors and methods for introducing nucleic acid into cells are discussed in available published literature.For example, the expression vector can be transferred into host cells by physical, chemical or biological means.See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).It is easy to understand that the introduction of an expression vector containing a polynucleotide encoding an antigen results in a pulsed cell.

[0110] The present invention includes various methods for pulsing APCs, including but not limited to loading APCs with peptide antigens, or cDNA or mRNA encoding peptide antigens. However, the present invention should not be construed as being limited to the particular form of antigen used to pulse APCs. Rather, the present invention encompasses other methods known in the art for generating antigen-loaded APCs. In one embodiment, APCs are transfected with mRNA encoding a defined antigen. mRNA corresponding to gene products of known sequence can be rapidly generated in vitro using appropriate primers and reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with a transcription reaction. Transfection of APCs with mRNA offers advantages over other antigen-loading techniques for generating pulsed APCs. For example, the ability to amplify RNA from microscopic amounts of tissue, i.e., tumor tissue, expands the use of APCs for vaccination to a large number of patients.

[0111] There are many methods that can be used to engineer DCs and other APCs, such as mRNA-based delivery, DNA plasmid-based delivery, etc., all of which are encompassed by the present invention, i.e., any delivery system can be used to engineer immune cells to express the mRAS peptides described herein.

[0112] It is understood that the antigenic compositions of the present invention may be produced by methods well known in the art, including, but not limited to, chemical synthesis by solid phase synthesis and purification from other products of chemical reactions by HPLC, or production by expression of a nucleic acid sequence (e.g., a DNA sequence) encoding a peptide antigen of the present invention in an in vitro translation system or in a living cell. Additionally, the antigenic composition may include cellular components isolated from a biological sample. The antigenic composition may be isolated and extensively dialyzed to remove one or more undesirable low molecular weight molecules, and / or lyophilized for easier formulation into a desired vehicle. Furthermore, it is understood that additional amino acids, mutations, chemical modifications, etc., made in the vaccine components, if any, do not substantially interfere with antibody recognition of the epitope sequence. The peptide sequences may be synthesized by methods known to those skilled in the art, such as, for example, peptide synthesis using an automated peptide synthesizer such as those available from Applied Biosystems, Inc. (Foster City, Calif.).

[0113] Longer peptides or polypeptides can also be prepared, e.g., by recombinant means. In certain embodiments, nucleic acids encoding the antigenic compositions and / or components described herein can be used to generate antigenic compositions in vitro or in vivo, e.g., for various compositions and methods of the invention. For example, in certain embodiments, the nucleic acid encoding the antigen is contained in a vector, e.g., in a recombinant cell. The nucleic acid can be expressed to produce a peptide or polypeptide that includes the antigenic sequence. The peptide or polypeptide can be secreted from the cell or can be part of or contained within the cell.

[0114] In one embodiment, an immune response may be promoted by transfecting or inoculating a mammal with a nucleic acid encoding an antigen. One or more cells contained within the target mammal then express the sequence encoded by the nucleic acid after administration of the nucleic acid to the mammal. The vaccine may also be in the form of, for example, a nucleic acid (e.g., cDNA or RNA) encoding all or part of the peptide or polypeptide sequence of the antigen. In vivo expression by the nucleic acid may be by, for example, a plasmid-type vector, a viral vector, or a viral / plasmid construct vector.

[0115] In another embodiment, the nucleic acid comprises a coding region that encodes all or part of a sequence encoding a suitable antigen, or an immunologically functional equivalent thereof. Of course, the nucleic acid may comprise and / or encode additional sequences, including, but not limited to, one or more immunomodulatory agents or adjuvants.

[0116] In certain embodiments, the immunological composition comprises immune cells stimulated by APCs loaded or pulsed with one or more mRAS peptide antigens described herein. For example, in one embodiment, the immunological composition comprises stimulated T cells cultured and activated with APCs loaded 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), which are then cultured and activated with APCs loaded 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, the naive cells and APCs are both derived from the same subject. In one embodiment, the naive cells and APCs are derived from different subjects within the same species.

[0117] Methods for detecting induction of cytotoxic T lymphocytes are well known. Foreign substances that enter the body are presented to T cells and B cells by the action of APPC. T cells that respond specifically to antigens presented by APCs are differentiated into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) by stimulation with the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as "activation" of T cells. Thus, CTL induction by epitopes of polypeptides or peptides or combinations thereof can be evaluated by presenting epitopes of polypeptides or peptides or combinations thereof to T cells by APCs and detecting induction of CTLs. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0118] The method for evaluating the induction effect of CTL using dendritic cells (DC) as APC is well known in the art. DC is a representative APC with strong CTL induction effect among APCs. In the method of the present invention, the epitope of the polypeptide or peptide or their combination is first expressed by DC, and then this DC is contacted with T cells. Detection of T cells with cytotoxic effect on the target cell after contact with DC indicates that the epitope of the polypeptide or peptide or their combination has the activity of inducing cytotoxic T cells. In addition, the induced immune response can also be examined by measuring the IFN-γ produced and released by CTL in the presence of antigen-presenting cells carrying immobilized peptide or peptide combination, by visualizing using anti-IFN-γ antibody such as ELISPOT assay.

[0119] Apart from 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, it has been shown that CTLs are induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0120] Antigens confirmed to have CTL-inducing activity by these methods are antigens that have DC activation effects and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity through antigen presentation by APCs can also be used as vaccines against antigen-associated disorders.

[0121] The induction of immunity by expression of mRAS peptide antigen can be further confirmed by observing the induction of antibody production against mRAS. For example, if antibodies against the antigen are induced in an experimental animal immunized with a composition encoding the antigen, and if the antigen-related pathology is suppressed by these antibodies, the composition is determined to induce immunity.

[0122] The induction of immunity by expression of the mRAS peptide antigen can be further confirmed by observing the induction of CD4+ T cells. Although CD4+ T cells can also lyse target cells, they primarily serve to induce other types of immune responses, such as the production of CTLs and antibodies. The types of CD4+ T cell help can be classified as Th1, Th2, Th9, Th17, T regulatory, or T follicular helper (T fh (CD4+ T cell subtypes can be characterized as CD4+ T cell subtypes. Each subtype of CD4+ T cell contributes to a specific type of immune response. In one embodiment, the composition selectively induces T follicular helper cells that drive a strong antibody response.

[0123] mRAS-specific TCR In some embodiments, the invention provides compositions comprising a polypeptide that specifically binds to RAS, mRAS, or a fragment thereof, hi 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.

[0124] In one embodiment, the TCR specifically binds to mRAS that contains a mutation relative to G12 of wild-type RAS. For example, in certain embodiments, the TCR specifically binds to mRAS that contains a G12C, G12D, G12R, or G12V mutation relative to wild-type RAS. In certain embodiments, the TCR specifically binds to a fragment of mRAS, where the fragment contains a mutation at the position corresponding to G12.

[0125] In some embodiments of the present invention, the TCR has antigen specificity for an mRAS peptide having a mutation at G12, as described above, and the mRAS peptide has any length. For example, the TCR may have antigen specificity for an mRAS peptide having a mutation corresponding to G12, an mRAS peptide having a length of about 8 to about 24 amino acid residues, or an mRAS peptide having a length of about 9 to about 11 amino acid residues. In one embodiment of the present invention, the TCR may have antigen specificity for an mRAS peptide having a mutation corresponding to G12, an 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 antigenic mRAS peptides with mutations corresponding to G12 that are specifically bound by the TCR can be found in Table 1.

[0126] In certain embodiments, the TCR specifically binds to an mRAS peptide in the context of a particular HLA molecule. The HLA molecules corresponding to the mRAS peptides can be found in Table 1.

[0127] HLA-A*02:01 G12C 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*02:01 molecule. For example, 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*02:01 molecule. C GV (SEQ ID NO: 1). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising the sequence KLVVVGA in the context of the HLA-A*02:01 molecule. C It specifically binds to the mRAS peptide containing GV (SEQ ID NO: 1).

[0128] HLA-A*02:01 G12D In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D 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 having a G12D mutation at a position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. D GV (SEQ ID NO: 2). For example, in one embodiment, the TCR specifically binds to an mRAS peptide that contains the sequence KLVVVGA in the context of the HLA-A*02:01 molecule. D It specifically binds to the mRAS peptide containing GV (SEQ ID NO:2).

[0129] HLA-A*02:01 G12R In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R 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 having a G12R mutation at a position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. RGV (SEQ ID NO: 3). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising the sequence KLVVVGA in the context of the HLA-A*02:01 molecule. R It specifically binds to the mRAS peptide containing GV (SEQ ID NO:3).

[0130] HLA-A*02:01 G12V In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V 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 having a G12V mutation at a position corresponding to RAS G12 in the context of an HLA-A*02:01 molecule. V GV (SEQ ID NO: 4). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising the sequence KLVVVGA in the context of the HLA-A*02:01 molecule. V It specifically binds to the mRAS peptide containing GV (SEQ ID NO: 4).

[0131] HLA-A*11:01 G12C 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*11:01 molecule. For example, 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*11:01 molecule. C GVGK (SEQ ID NO: 5) or VVVGA C GVGK (SEQ ID NO: 6). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*11:01 molecule. C GVGK (SEQ ID NO: 5) or VVVGA C It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO:6).

[0132] HLA-A*11:01 G12D In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a 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 having a G12D mutation at a position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*11:01 molecule. D GVGK (SEQ ID NO: 7) or VVVGA D It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO:8).

[0133] HLA-A*11:01 G12R In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a 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 having a G12R mutation at a position corresponding to RAS G12 in the context of an HLA-A*11:01 molecule. R GVGK (SEQ ID NO: 9) or VVVGA R GVGK (SEQ ID NO: 10). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*11:01 molecule. R GVGK (SEQ ID NO: 9) or VVVGA R It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO: 10).

[0134] HLA-A*11:01 G12V In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12 in the context of the HLA-A*11:01 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12 in the context of the HLA-A*11:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V GVGK (SEQ ID NO: 12). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*11:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO: 12).

[0135] HLA-A*03:01 G12C 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*03:01 molecule. For example, 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*03:01 molecule. C GVGK (SEQ ID NO: 5) or VVVGA C GVGK (SEQ ID NO: 6). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*03:01 molecule. C GVGK (SEQ ID NO: 5) or VVVGA C It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO:6).

[0136] HLA-A*03:01 G12D In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a 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 having a G12D mutation at a position corresponding to RAS G12 in the context of an HLA-A*03:01 molecule. D GVGK (SEQ ID NO: 7) or VVVGA D GVGK (SEQ ID NO: 8). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*03:01 molecule. D GVGK (SEQ ID NO: 7) or VVVGA D It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO:8).

[0137] HLA-A*03:01 G12R In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a 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 having a G12R mutation at a position corresponding to RAS G12 in the context of an HLA-A*03:01 molecule. R GVGK (SEQ ID NO: 9) or VVVGA R GVGK (SEQ ID NO: 10). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*03:01 molecule. R GVGK (SEQ ID NO: 9) or VVVGA R It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO: 10).

[0138] HLA-A*03:01 G12V In one embodiment, the TCR specifically binds to an mRAS peptide having a G12G mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12G mutation at a 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 having a G12G mutation at a position corresponding to RAS G12 in the context of an HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V GVGK (SEQ ID NO: 12). For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising VVGA in the context of the HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V It specifically binds to the mRAS peptide containing GVGK (SEQ ID NO: 12).

[0139] HLA-B*07:02 G12C 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 the HLA-B*07:02 molecule. For example, 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 the HLA-B*07:02 molecule. C For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 13) in the context of the HLA-B*07:02 molecule. C It specifically binds to the mRAS peptide containing GVGKSAL (SEQ ID NO: 13).

[0140] HLA-B*07:02 G12D In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide having a G12D mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. DFor example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 14) in the context of the HLA-B*07:02 molecule. D It specifically binds to the mRAS peptide containing GVGKSAL (SEQ ID NO: 14).

[0141] HLA-B*07:02 G12R In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. R For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15) in the context of the HLA-B*07:02 molecule. R It specifically binds to the mRAS peptide containing GVGKSAL (SEQ ID NO: 15).

[0142] HLA-B*07:02 G12V In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12. In one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. For example, in one embodiment, the TCR specifically binds to an mRAS peptide having a G12V mutation at a position corresponding to RAS G12 in the context of the HLA-B*07:02 molecule. V For example, in one embodiment, the TCR specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 16) in the context of the HLA-B*07:02 molecule. V It specifically binds to the mRAS peptide containing GVGKSAL (SEQ ID NO: 16).

[0143] TCR831 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0144] In one 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 39 (TRAV39-01*01; also referred to herein as "TRAV39") CDR1. In one 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, where the TRAV39 CDR1 comprises the amino acid sequence of STTSDRL (SEQ ID NO: 17).

[0145] In one 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 one 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, where the TRAV39 CDR2 comprises the amino acid sequence of VLLSNGAVK (SEQ ID NO: 18).

[0146] In one 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 one 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, where the TRAV39 CDR3 comprises the amino acid sequence of CAVDKDGGYQKVTF (SEQ ID NO: 19).

[0147] In one 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.

[0148] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV39. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV39, wherein the variable region of TRAV39 comprises the amino acid sequence of ELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAVHDLSATYFCAVDKDGGYQKVTFGTGTKLQVIP (SEQ ID NO: 20).

[0149] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 21).

[0150] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSLTLHITAAVHDLSATYFCAVDKDGGYQKVTFGTGTKLQVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 22).

[0151] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0152] In one 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 beta variable 20-1 (TRBV20-01*01; also referred to herein as "TRBV20-1") CDR1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR1, where the TRBV20-1 CDR1 comprises the amino acid sequence of LDFQATTM (SEQ ID NO:23).

[0153] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR2. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR2, where the TRBV20-1 CDR2 comprises the amino acid sequence of TSNEGSKAT (SEQ ID NO: 24).

[0154] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR3. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV20-1 CDR3, where the TRBV20-1 CDR3 comprises the amino acid sequence of CSASPRAGQLSSYNSPLHF (SEQ ID NO: 25).

[0155] In one embodiment, a TCR that specifically binds to an 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.

[0156] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRBV20-1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRBV20-1, wherein the variable region of TRBV20-1 comprises the amino acid sequence of GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASPRAGQLSSYNSPLHFGNGTRLTV (SEQ ID NO: 26).

[0157] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 27).

[0158] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence of MLLLLLLLGPGISLLLPGSLAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASPRAGQLSSYNSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO:28).

[0159] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR beta 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 alpha chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR beta 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 relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 has the sequence VVVGA in the context of the HLA-A*11:01 molecule. VIn one embodiment, the TCR comprises (a) a TCR α 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 GVGK (SEQ ID NO: 12) in the context of the HLA-A*11:01 molecule. C It binds to mRAS peptides containing GVGK (SEQ ID NO:6).

[0160] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR beta chain comprising TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR beta chain comprising TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3, and binds to an mRAS peptide comprising a G12V or G12C mutation relative to RAS G12. In one embodiment, the antibody comprises (a) a TCR α 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 has the sequence VVVGA in the context of the HLA-A*11:01 molecule. V In one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR α chain comprising TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3, and (b) a TCR β chain comprising TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3, and has the sequence VVVGA in the context of the HLA-A*11:01 molecule. C It binds to mRAS peptides containing GVGK (SEQ ID NO:6).

[0161] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. V and TRBV20-1-CDR3: SEQ ID NO:25, or a variant or variant thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. CBinds to mRAS peptides containing GVGK (SEQ ID NO:6): 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 a variant or variants thereof.

[0162] In one embodiment, a 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: 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 a variant or variants thereof. In one embodiment, the TCR comprises all 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 a variant or variants thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. Vand TRBV20-1-CDR3: SEQ ID NO:25, or variants or variants thereof. The TCR comprises all CDRs selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. C Binds to mRAS peptides containing GVGK (SEQ ID NO:6): 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 a variant or variants thereof.

[0163] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 29).

[0164] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:30).

[0165] TCR833 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0166] In one 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 12-1 (TRAV12-1*01; also referred to herein as "TRAV12-1") CDR1. In one 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, where the TRAV12-1 CDR1 comprises the amino acid sequence of SNSASQSF (SEQ ID NO:31).

[0167] In one 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 one 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, where the TRAV12-1 CDR2 comprises the amino acid sequence of SVYSSGNE (SEQ ID NO: 32).

[0168] In one 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 one 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 amino acid sequence of CAVNPPDTGFQKLVF (SEQ ID NO: 33).

[0169] In one 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.

[0170] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV12-1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV12-1, wherein the variable region of TRAV12-1 comprises the amino acid sequence of RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVNPPDTGFQKLVFGTGTRLLVSP (SEQ ID NO: 34).

[0171] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 35).

[0172] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence of MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCAVNPPDTGFQKLVFGTGTRLLVSPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 36).

[0173] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0174] In one 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 beta variable 28 (TRBV28*01; also referred to herein as "TRBV28") CDR1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR1, where the TRBV28 CDR1 comprises the amino acid sequence of DMDHENM (SEQ ID NO: 37).

[0175] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR2. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR2, where the TRBV28 CDR2 comprises the amino acid sequence of FSYDVKME (SEQ ID NO: 38).

[0176] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR3. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV28 CDR3, where the TRBV28 CDR3 comprises the amino acid sequence of CASSLSFRQGLREQYF (SEQ ID NO: 39).

[0177] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3.

[0178] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV28. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV28, wherein the variable region of TRBV28 comprises the amino acid sequence of MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSFRQGLREQYFGPGTRLTVT (SEQ ID NO: 40).

[0179] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 41).

[0180] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSFRQGLREQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:42).

[0181] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR beta chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR beta chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds to an mRAS peptide comprising a G12V or G12C mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 has the sequence VVVGA in the context of the HLA-A*11:01 molecule. VIn one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR α 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 has the sequence VVVGA in the context of the HLA-A*11:01 molecule. C It binds to mRAS peptides containing GVGK (SEQ ID NO:6).

[0182] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR beta chain comprising TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR beta chain comprising TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and binds to an mRAS peptide comprising a G12V or G12C mutation relative to RAS G12. In one embodiment, the antibody comprises (a) a TCR α chain comprising a TRAV12-1 CDR1, a TRAV12-1 CDR2, and a TRAV12-1 CDR3, and (b) a TCR β chain comprising a TRBV28 CDR1, a TRBV28 CDR2, and a TRBV28 CDR3, and has the sequence VVVGA in the context of the HLA-A*11:01 molecule. V In one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 12). In one embodiment, the TCR comprises (a) a TCR α chain comprising TRAV12-1 CDR1, TRAV12-1 CDR2, and TRAV12-1 CDR3, and (b) a TCR β chain comprising TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3, and has the sequence VVVGA in the context of the HLA-A*11:01 molecule. C It binds to mRAS peptides containing GVGK (SEQ ID NO:6).

[0183] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. V and TRBV28-CDR3: SEQ ID NO:39, or a variant or variant thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. CBinds to mRAS peptides containing GVGK (SEQ ID NO:6): 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 a variant or variants thereof.

[0184] In one embodiment, a 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: 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 a variant or variants thereof. In one embodiment, the TCR comprises all 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 a variant or variants thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. Vand TRBV28-CDR3: SEQ ID NO:39, or a variant or variant thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: VVVGA in the context of the HLA-A*11:01 molecule. C Binds to mRAS peptides containing GVGK (SEQ ID NO:6): 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 a variant or variants thereof.

[0185] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 43).

[0186] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:44).

[0187] TCR897 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0188] In one 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 17 (TRAV17) CDR1. In one 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 amino acid sequence of KTSINNL (SEQ ID NO: 45).

[0189] In one 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 one 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, where the TRAV17 CDR2 comprises the amino acid sequence of LIRSNEREK (SEQ ID NO: 46).

[0190] In one 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 one 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, where the TRAV17 CDR3 comprises the amino acid sequence of CATDPGGFKTIF (SEQ ID NO: 47).

[0191] In one 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.

[0192] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV17. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV17, wherein the variable region of TRAV17 comprises the amino acid sequence of SQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATD (SEQ ID NO: 169).

[0193] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 48).

[0194] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:49).

[0195] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0196] In one 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 beta variable 11-2 (TRBV11-2) CDR1. In one 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, where the TRBV11-2 CDR1 comprises the amino acid sequence of ISGHATL (SEQ ID NO:50).

[0197] In one 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 one 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, where the TRBV11-2 CDR2 comprises the amino acid sequence of QFQNNGVV (SEQ ID NO:51).

[0198] In one 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 one 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, where the TRBV11-2 CDR3 comprises the amino acid sequence of CASSLYGGSISYEQYF (SEQ ID NO:52).

[0199] In one 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.

[0200] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV11-2. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV11-2, wherein the variable region of TRBV11-2 comprises the amino acid sequence of EAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSL (SEQ ID NO: 170).

[0201] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 53).

[0202] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLYGGSISYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:54).

[0203] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta 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 alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta 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 relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 has the VVGA domain in the context of the HLA-A*11:01 molecule. VIn one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 11). In one embodiment, the TCR comprises (a) a TCR α 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 GVGK (SEQ ID NO: 11) in the context of an HLA-A*11:01 molecule. C In one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises (a) a TCR α 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 GVGK (SEQ ID NO: 5) in the context of an HLA-A*11:01 molecule. D In one embodiment, the TCR comprises (a) a TCR α 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 GVGK (SEQ ID NO: 7) in the context of an HLA-A*11:01 molecule. R It binds to mRAS peptides containing GVGK (SEQ ID NO:9).

[0204] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta chain comprising TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta chain comprising TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3, and binds to an mRAS peptide comprising a G12V, G12C, or G12D mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 has the VVGA domain in the context of the HLA-A*11:01 molecule. V In one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 11). In one embodiment, the TCR comprises (a) a TCR α 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 to an mRAS peptide comprising GVGK (SEQ ID NO: 11) in the context of the HLA-A*11:01 molecule. C In one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 5). In one embodiment, the TCR comprises (a) a TCR α 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 to an mRAS peptide comprising GVGK (SEQ ID NO: 5) in the context of the HLA-A*11:01 molecule. DIn one embodiment, the TCR binds to an mRAS peptide comprising GVGK (SEQ ID NO: 7). In one embodiment, the TCR comprises (a) a TCR α 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 to an mRAS peptide comprising GVGK (SEQ ID NO: 7) in the context of the HLA-A*11:01 molecule. R It binds to mRAS peptides containing GVGK (SEQ ID NO:9).

[0205] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one CDR selected from the group consisting of: TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:50; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:37; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV17-CDR1: SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 50; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1: SEQ ID NO: 37; TRBV11-2-CDR2: SEQ ID NO: 51; and TRBV11-2-CDR3: SEQ ID NO: 52, or a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVGA in the context of the HLA-A*11:01 molecule. Vand TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVGA in the context of the HLA-A*11:01 molecule. C and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVGA in the context of the HLA-A*11:01 molecule. D and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVGA in the context of the HLA-A*11:01 molecule. R Binds to mRAS peptides containing GVGK (SEQ ID NO:9): TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:50; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:37; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variants thereof.

[0206] In one embodiment, a 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:50; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:37; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variants thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of the following and binds to an mRAS peptide containing a G12V, G12C, or G12D mutation relative to RAS G12: TRAV17-CDR1: SEQ ID NO: 45; TRAV17-CDR2: SEQ ID NO: 50; TRAV17-CDR3: SEQ ID NO: 47; TRBV11-2-CDR1: SEQ ID NO: 37; TRBV11-2-CDR2: SEQ ID NO: 51; and TRBV11-2-CDR3: SEQ ID NO: 52, or a variant or variants thereof. ... V and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: Cand TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: D and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variant thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of: R Binds to mRAS peptides containing GVGK (SEQ ID NO:9): TRAV17-CDR1: SEQ ID NO:45; TRAV17-CDR2: SEQ ID NO:50; TRAV17-CDR3: SEQ ID NO:47; TRBV11-2-CDR1: SEQ ID NO:37; TRBV11-2-CDR2: SEQ ID NO:51; and TRBV11-2-CDR3: SEQ ID NO:52, or a variant or variants thereof.

[0207] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 55).

[0208] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:56).

[0209] TCR896 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0210] In one 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 19 (TRAV19) CDR1. In one embodiment, a 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 amino acid sequence of ETRDTTYYL (SEQ ID NO:57).

[0211] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR2. In one embodiment, a 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 amino acid sequence of RRNSFDEQNE (SEQ ID NO:58).

[0212] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRAV19 CDR3. In one embodiment, a 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 amino acid sequence of CALSEAGTYKYIF (SEQ ID NO:59).

[0213] In one embodiment, a 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.

[0214] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV19. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV19, wherein the variable region of TRAV19 comprises the amino acid sequence of AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSE (SEQ ID NO: 171).

[0215] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 60).

[0216] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 61).

[0217] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0218] In one 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 beta variable 9 (TRBV9) CDR1. In one embodiment, a 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 amino acid sequence of RSGDLSV (SEQ ID NO: 62).

[0219] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR2. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR2, where the TRBV9 CDR2 comprises the amino acid sequence of QYYNGEER (SEQ ID NO: 63).

[0220] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR3. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV9 CDR3, where the TRBV9 CDR3 comprises the amino acid sequence of CASSVAGGGQETQYF (SEQ ID NO: 64).

[0221] In one embodiment, a 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.

[0222] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV9. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV9, wherein the variable region of TRBV9 comprises the amino acid sequence of DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSV (SEQ ID NO: 172).

[0223] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 65).

[0224] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVAGGGQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 66).

[0225] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR beta chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR beta chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising a G12V mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 has the VVGA domain in the context of the HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V It binds to mRAS peptides containing GVGK (SEQ ID NO:12).

[0226] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR beta chain comprising TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR beta chain comprising TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and binds to an mRAS peptide comprising a G12V mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α chain comprising TRAV19 CDR1, TRAV19 CDR2, and TRAV19 CDR3, and (b) a TCR β chain comprising TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3, and has the VVGA domain in the context of the HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V It binds to mRAS peptides containing GVGK (SEQ ID NO:12).

[0227] In one embodiment, a TCR that specifically binds to an mRAS peptide having 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 a variant or variants 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: VVGA in the context of the HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V Binds to mRAS peptides containing GVGK (SEQ ID NO:12): 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 a variant or variants thereof.

[0228] In another embodiment, a 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: 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 a variant or variants thereof. In one embodiment, the TCR comprises all CDRs 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 a variant or variant thereof. ... VVGA in the context of the HLA-A*03:01 molecule. V GVGK (SEQ ID NO: 11) or VVVGA V Binds to mRAS peptides containing GVGK (SEQ ID NO:12): 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 a variant or variants thereof.

[0229] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 67).

[0230] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:68).

[0231] TCR847 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0232] In one 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 17 (TRAV17) CDR1. In one 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, where the TRAV17 CDR1 comprises the amino acid sequence of KTSINNL (SEQ ID NO: 69).

[0233] In one 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 one 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, where the TRAV17 CDR2 comprises the amino acid sequence of LIRSNEREK (SEQ ID NO: 70).

[0234] In one 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 one 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, where the TRAV17 CDR3 comprises the amino acid sequence of CATFPNFGNEKLTF (SEQ ID NO: 71).

[0235] In one 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.

[0236] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV17. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV17, wherein the variable region of TRAV17 comprises the amino acid sequence of SQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATF (SEQ ID NO: 173).

[0237] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 72).

[0238] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATFPNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:73).

[0239] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0240] In one 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 beta variable 10-3 (TRBV10-3) CDR1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR1, where the TRBV10-3 CDR1 comprises the amino acid sequence of TENHRYM (SEQ ID NO: 74).

[0241] In one embodiment, a 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 one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR2, where the TRBV10-3 CDR2 comprises the amino acid sequence of YSYGVKDT (SEQ ID NO: 75).

[0242] In one embodiment, a 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 one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV10-3 CDR3, where the TRBV10-3 CDR3 comprises the amino acid sequence of CAISESERYYEQYF (SEQ ID NO: 76).

[0243] In one embodiment, a 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.

[0244] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV10-3. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV10-3, wherein the variable region of TRBV10-3 comprises the amino acid sequence of DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISE (SEQ ID NO: 174).

[0245] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 77).

[0246] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISESERYYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:78).

[0247] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta 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 relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 is GA in the context of the HLA-B*07:02 molecule. R It binds to an mRAS peptide containing GVGKSAL (SEQ ID NO: 15).

[0248] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta chain comprising TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3, and (b) a TCR beta chain comprising TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3, and binds to an mRAS peptide comprising a G12R mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α chain comprising a TRAV17 CDR1, a TRAV17 CDR2, and a TRAV17 CDR3, and (b) a TCR β chain comprising a TRBV10-3 CDR1, a TRBV10-3 CDR2, and a TRBV10-3 CDR3, and is GA in the context of the HLA-B*07:02 molecule. R It binds to an mRAS peptide containing GVGKSAL (SEQ ID NO: 15).

[0249] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR selected from the group consisting of: TRAV17-CDR2: SEQ ID NO:76; TRAV17-CDR3: SEQ ID NO:77; TRAV17-CDR4: SEQ ID NO:78; TRAV17-CDR5: SEQ ID NO:79; TRAV17-CDR6: SEQ ID NO:80; TRAV17-CDR7: SEQ ID NO:81; TRAV17-CDR8: SEQ ID NO:82; R Binds to mRAS peptides containing GVGKSAL (SEQ ID NO:15): 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 a variant or variants thereof.

[0250] In another embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 comprises all 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 a variant or variants thereof. In one embodiment, the TCR comprises all 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 a variant or variants thereof. ... R Binds to mRAS peptides containing GVGKSAL (SEQ ID NO:15): 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 a variant or variants thereof.

[0251] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 79).

[0252] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:80).

[0253] TCR864 In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR α chain.

[0254] In one 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 4 (TRAV4) CDR1. In one 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, wherein the TRAV4 CDR1 comprises the amino acid sequence of NNIATNDYI (SEQ ID NO: 81).

[0255] In one 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 one 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, where the TRAV4 CDR2 comprises the amino acid sequence of QGYKTKV (SEQ ID NO: 82).

[0256] In one 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 one 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 amino acid sequence of CLVGDFNSNSGYALNF (SEQ ID NO: 83).

[0257] In one 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.

[0258] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV4. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises the variable region of TRAV4, wherein the variable region of TRAV4 comprises the amino acid sequence of LAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGD (SEQ ID NO: 175).

[0259] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 84).

[0260] In one embodiment, the TCR comprises a TCR alpha chain comprising the amino acid sequence MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDFNSNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 85).

[0261] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises one or more of the following: CDR1, CDR2, and CDR3 of the TCR β chain.

[0262] In one 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 beta variable 7-2 (TRBV7-2) CDR1. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR1, where the TRBV7-2 CDR1 comprises the amino acid sequence of ISGHTAL (SEQ ID NO: 86).

[0263] In one embodiment, a 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 one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR2, where the TRBV7-2 CDR2 comprises the amino acid sequence of YFQGNSAP (SEQ ID NO: 87).

[0264] In one embodiment, a 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 one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a TRBV7-2 CDR3, where the TRBV7-2 CDR3 comprises the amino acid sequence of CASKVYGYTF (SEQ ID NO: 88).

[0265] In one embodiment, a 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.

[0266] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV7-2. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12 mutation at a position corresponding to RAS G12 comprises a variable region of TRBV7-2, wherein the variable region of TRBV7-2 comprises the amino acid sequence of GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASK (SEQ ID NO: 176).

[0267] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises a constant region. In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at a position corresponding to RAS G12 comprises a constant region, wherein the constant region comprises the amino acid sequence of EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 89).

[0268] In one embodiment, the TCR comprises a TCR beta chain comprising the amino acid sequence MGTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASKVYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 90).

[0269] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR beta chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising one or more of TRAV4 CDR1, TRAV4 CDR2, and TRAV4 CDR3, and (b) a TCR beta 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 relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α 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 is GA in the context of the HLA-B*07:02 molecule. R It binds to an mRAS peptide containing GVGKSAL (SEQ ID NO: 15).

[0270] In one embodiment, the TCR comprises (a) a TCR alpha chain comprising a TRAV4 CDR1, a TRAV4 CDR2, and a TRAV4 CDR3, and (b) a TCR beta chain comprising a TRBV7-2 CDR1, a TRBV7-2 CDR2, and a TRBV7-2 CDR3. In one embodiment, the TCR comprises (a) a TCR alpha chain comprising a TRAV4 CDR1, a TRAV4 CDR2, and a TRAV4 CDR3, and (b) a TCR beta chain comprising a TRBV7-2 CDR1, a TRBV7-2 CDR2, and a TRBV7-2 CDR3, and binds to an mRAS peptide comprising a G12R mutation relative to RAS G12. In one embodiment, the TCR comprises (a) a TCR α chain comprising a TRAV4 CDR1, a TRAV4 CDR2, and a TRAV4 CDR3, and (b) a TCR β chain comprising a TRBV7-2 CDR1, a TRBV7-2 CDR2, and a TRBV7-2 CDR3, and is GA in the context of the HLA-B*07:02 molecule. R It binds to an mRAS peptide containing GVGKSAL (SEQ ID NO: 15).

[0271] In one embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 comprises at least one CDR 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 a variant or variants thereof. In one embodiment, the TCR comprises at least one CDR 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 a variant or variants thereof. ... R Binds to mRAS peptides containing GVGKSAL (SEQ ID NO:15): 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 a variant or variants thereof.

[0272] In another embodiment, a TCR that specifically binds to an mRAS peptide having a G12R mutation at the position corresponding to RAS G12 comprises all 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 a variant or variants thereof. In one embodiment, the TCR comprises all CDRs selected from the group consisting of the following and binds to an mRAS peptide comprising a G12R mutation relative to RAS G12: 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 a variant or variants thereof. ... R Binds to mRAS peptides containing GVGKSAL (SEQ ID NO:15): 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 a variant or variants thereof.

[0273] In one embodiment, the composition comprises a fusion protein comprising the TCR alpha and beta chains described above. In one embodiment, the fusion protein comprises a linker domain separating the TCR alpha and beta chains. 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, GSG-T2A comprises the amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 91).

[0274] In one embodiment, the composition comprises a fusion protein comprising the following amino acid sequence: (SEQ ID NO:92).

[0275] In certain embodiments, the composition comprises a fusion protein comprising a linker domain separating the TCR alpha and beta chains. In one embodiment, the linker domain is a cleavable linker domain. Any suitable linker domain can be used such that the function of the alpha and beta chains is maintained.

[0276] In certain embodiments, the composition comprises a peptide or polypeptide (e.g., mRAS peptide antigen or TCR) that comprises an amino acid sequence that is substantially homologous to the amino acid sequence of the mRAS peptide, 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 a degree of identity of 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% to the original amino acid sequence.

[0277] In some embodiments, the composition comprises 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 peptides having mutations, such as point mutations, in the amino acid sequence of an mRAS peptide or TCR described herein, or a portion thereof.

[0278] In some embodiments, the TCR comprises an amino acid sequence having at least about 85% amino acid identity to one or more CDR sequences described herein. The invention 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.

[0279] In one embodiment, the composition comprises a polypeptide having a CDR sequence at least about 85% identical to a CDR sequence set forth herein. The invention 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 a CDR sequence set forth herein.

[0280] The peptides of the present invention can be produced 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 performed, for example, using an ABI 431 A peptide synthesizer (Perkin Elmer) following the instructions provided by the manufacturer. Alternatively, peptides can be produced by recombinant means or by cleavage from a longer polypeptide. The composition of the peptide can be confirmed by amino acid analysis or sequencing.

[0281] Variants of the polypeptides according to the invention may be (i) those in which one or more amino acid residues have been replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), where such replaced amino acid residue may or may not be one encoded by the genetic code, (ii) those in which one or more modified amino acid residues are present, e.g., residues modified by attachment of a substituent group, (iii) those in which the polypeptide is an alternative splicing variant of the polypeptide of the invention, (iv) fragments of the polypeptides, and / or (v) those in which the polypeptide is fused to another polypeptide, such as a leader or secretion sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teachings herein.

[0282] As known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutions of one polypeptide with the sequence of a second polypeptide. A variant is a polypeptide sequence that differs from the original sequence, preferably differs from the original sequence by less than 40% of the residues per segment of interest, more preferably differs from the original sequence by less than 25% of the residues per segment of interest, more preferably differs from the original sequence by less than 10% of the residues per segment of interest, and most preferably, a variant is defined as including a polypeptide sequence that differs from the original protein sequence by only a few residues per segment of interest, while at the same time being sufficiently homologous to the original sequence to preserve the functionality of the original sequence and / or its ability to bind to ubiquitin or ubiquitinated proteins. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% homologous or identical to the original amino acid sequence. The degree of identity between two polypeptides is determined using computer algorithms and methods that are well known to those skilled in the art. The 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)].

[0283] The polypeptide of the present invention can be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation are studied by adding dog microsomal membranes or Xenopus egg extracts (US Pat. No. 6,103,489) to standard translation reactions.

[0284] The polypeptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing the unnatural amino acid during translation. Various approaches are available for introducing unnatural amino acids during protein translation. As an example, special tRNAs, such as tRNAs with suppressor properties, suppressor tRNAs, have been used in the process of site-specific unnatural amino acid substitution (SNAAR). SNAARs require a unique codon in the mRNA and suppressor tRNA, which acts to target the unnatural amino acid to a unique site during protein synthesis (described in WO 90 / 05785). However, the suppressor tRNA must not be recognized by the aminoacyl-tRNA synthetase present in the protein translation system. In some cases, unnatural amino acids can be formed after the tRNA molecule is aminoacylated using chemical reactions that specifically modify natural amino acids and do not significantly change the functional activity of the aminoacylated tRNA. These reactions are called post-aminoacylation modifications. For example, the cognate tRNA (tRNA LYS The epsilon amino group of the lysine bound to α-amino-2,4-diamino-1-propanediol can be modified with an amine-specific photoaffinity label.

[0285] The peptides of the present invention can be converted into pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or 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 toluenesulfonic acid, and the like.

[0286] nucleic acid molecule In one embodiment, the present invention provides a composition comprising an isolated nucleic acid molecule encoding one or more of the peptides or polypeptides described herein. For example, in certain embodiments, the composition comprises DNA, RNA, mRNA, or cDNA encoding one or more of the peptides or polypeptides described herein.

[0287] In one embodiment, a composition comprises one or more isolated nucleic acid molecules encoding one or more antigenic mRAS peptides described herein. For example, in one embodiment, a composition comprises one or more isolated nucleic acid molecules encoding one or more antigenic mRAS peptides comprising an amino acid sequence selected from SEQ ID NOs:1-16.

[0288] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence selected from SEQ ID NOs: 1-92. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having substantial homology to an amino acid sequence selected from 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 SEQ ID NOs: 1-92. In certain embodiments, the nucleic acid molecule comprises 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 ten or more mutations, e.g., point mutations, compared to an amino acid sequence selected from SEQ ID NOs: 1-92.

[0289] In one embodiment, a composition comprises an isolated nucleic acid molecule 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.

[0290] A nucleic acid molecule encoding TCR831 In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV39 CDR1, TRAV39 CDR2, and TRAV39 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV39 CDR1 comprising the amino acid sequence of SEQ ID NO:17, TRAV39 CDR2 comprising the amino acid sequence of SEQ ID NO:18, and TRAV39 CDR3 comprising the amino acid sequence of SEQ ID NO:19. In one embodiment, the nucleic acid sequence encoding TRAV39 CDR1 comprises ACCACTTCAGA (SEQ ID NO:93). In one embodiment, the nucleic acid sequence encoding TRAV39 CDR2 comprises TTGCTATCAAATGGAGCAGTG (SEQ ID NO:94). In one embodiment, the nucleic acid sequence encoding TRAV39 CDR3 comprises GCCGTGGCAAGGATGGGGGTTACC (SEQ ID NO:95).

[0291] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising the variable region of TRAV39 comprising the amino acid sequence of SEQ ID NO:20. In one embodiment, the nucleic acid sequence encoding the variable region of TRAV39 comprises ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTAAGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGACAGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGATTAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCACATCACAGCTGCCGTGCATGACCTCTCTGCCACCTACTTCTGTGCCGTGGACAAGGATGGGGGTTACCAGAAAGTTACCTTTGGAACTGGAACAAAGCTCCAAGTCATCCCAA (SEQ ID NO: 96).

[0292] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:21. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 97).

[0293] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 98).

[0294] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β 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 the TRBV20-1 CDR3 comprises AGTGCTAGCCCACGGGCGGGACAGTTGAGCTCCTATAATTCACCCCTCCAC (SEQ ID NO: 101).

[0295] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising a variable region of TRBV20-1 comprising the amino acid sequence of SEQ ID NO:26. In one embodiment, the nucleic acid sequence encoding the variable region of TRBV20-1 comprises ATGCTGCTGCTTCTTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTACCTGGGAGCTTGGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGCCCACGGGCGGGACAGTTGAGCTCCTATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGAC (SEQ ID NO: 102).

[0296] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 103).

[0297] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO: 28. In one embodiment, the nucleic acid sequence encoding the TCR β chain comprisesIt contains ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTACCTGGGAGCTTGGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGCCCACGGGCGGGACAGTTGAGCTCCTATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA (SEQ ID NO: 104).

[0298] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0299]

[0300] A nucleic acid molecule encoding TCR833 In an embodiment, 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 an embodiment, 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 AACAGTGCTTCTCAGTC (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 the TRAV12-1 CDR3 comprises GCGGTGAACCCCCCGGACACAGGCTTTCAGAAACTTGTA (SEQ ID NO: 109).

[0301] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising the variable region of TRAV12-1 comprising the amino acid sequence of SEQ ID NO:34. In one embodiment, the nucleic acid sequence encoding the variable region of TRAV12-1 comprises ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAACGAAGATGGAAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGCGGTGAACCCCCCGGACACAGGCTTTCAGAAACTTGTATTTGGAACTGGCACCCGACTTCTGGTCAGTCCAA (SEQ ID NO: 110).

[0302] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:35. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 111).

[0303] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 36. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 112).

[0304] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV28 CDR1 comprising the amino acid sequence of SEQ ID NO:37, TRBV28 CDR2 comprising the amino acid sequence of SEQ ID NO:38, and TRBV28 CDR3 comprising the amino acid sequence of SEQ ID NO:39. In one embodiment, the nucleic acid sequence encoding TRBV28 CDR1 comprises ATGGACCATGAAAAT (SEQ ID NO:113). In one embodiment, the nucleic acid sequence encoding TRBV28 CDR2 comprises TCATATGATGTTAAAATG (SEQ ID NO:114). In one embodiment, the nucleic acid sequence encoding TRBV28 CDR3 comprises GCCAGCAGTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTAC (SEQ ID NO:115).

[0305] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising a variable region of TRBV28 comprising the amino acid sequence of SEQ ID NO:40. In one embodiment, the nucleic acid sequence encoding the variable region of TRBV28 comprises ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCCAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACA (SEQ ID NO: 116).

[0306] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 41. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 117).

[0307] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO:42.In one embodiment, the nucleic acid sequence encoding the TCRβ chain comprises ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCCAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATCCTTCCGGCAGGGCCTTCGCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 118).

[0308] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0309]

[0310] A nucleic acid molecule encoding TCR897 In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV17 CDR1 comprising the amino acid sequence of SEQ ID NO:45, TRAV17 CDR2 comprising the amino acid sequence of SEQ ID NO:46, and TRAV17 CDR3 comprising the amino acid sequence of SEQ ID NO:47. In one embodiment, the nucleic acid sequence encoding TRAV17 CDR1 comprises ACTAGTATAAACAAT (SEQ ID NO:121). In one embodiment, the nucleic acid sequence encoding TRAV17 CDR2 comprises ATACGTTCAAATGAAAGAGAG (SEQ ID NO:122). In one embodiment, the nucleic acid sequence encoding TRAV17 CDR3 comprises TGTGCTACGGACCCTGGAGGCTTCAAAACTATCTTT (SEQ ID NO:123).

[0311] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:48. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 124).

[0312] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 49. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 125).

[0313] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β 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 the TRBV11-2 CDR3 comprises TGTGCCAGCAGCTTATATGGGGGGTCGATCTCCTACGAGCAGTACTTC (SEQ ID NO: 128).

[0314] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 129).

[0315] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO:54.In one embodiment, the nucleic acid sequence encoding the TCRβ chain comprises ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCAGAACTCACAGAAGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATAGAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCATGCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTTCTGATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCTAAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACTCCACTCTCAAGATCCAGCCTGCAAAGCTTGAGGACTCGGCCGTGTATCTCTGTGCCAGCAGCTTATATGGGGGGTCGATCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 130).

[0316] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0317]

[0318] A nucleic acid molecule encoding TCR896 In an embodiment, 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 an embodiment, 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).

[0319] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:60. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 136).

[0320] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 61. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 137).

[0321] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV9 CDR1 comprising the amino acid sequence of SEQ ID NO:62, TRBV9 CDR2 comprising the amino acid sequence of SEQ ID NO:63, and TRBV9 CDR3 comprising the amino acid sequence of SEQ ID NO:64. In one embodiment, the nucleic acid sequence encoding TRBV9 CDR1 comprises TCTGGAGACCTCTCT (SEQ ID NO:138). In one embodiment, the nucleic acid sequence encoding TRBV9 CDR2 comprises CGGAACTCTTTTGATGAGCAAAAT (SEQ ID NO:139). In one embodiment, the nucleic acid sequence encoding TRBV9 CDR3 comprises TGTGCTCTGAGTGAGGCAGGAACCTACAAATACATCTTT (SEQ ID NO:140).

[0322] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 65. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 141).

[0323] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO:66.In one embodiment, the nucleic acid sequence encoding the TCRβ chain comprises ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGCTGGGGGGGGACAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 142).

[0324] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0325]

[0326] A nucleic acid molecule encoding TCR847 In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV17 CDR1, TRAV17 CDR2, and TRAV17 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising one or more of TRAV17 CDR1 comprising the amino acid sequence of SEQ ID NO:69, TRAV17 CDR2 comprising the amino acid sequence of SEQ ID NO:70, and TRAV17 CDR3 comprising the amino acid sequence of SEQ ID NO:71. In one embodiment, the nucleic acid sequence encoding TRAV17 CDR1 comprises ACTAGTATAAACAAT (SEQ ID NO:145). In one embodiment, the nucleic acid sequence encoding TRAV17 CDR2 comprises ATACGTTCAAATGAAAGAGAG (SEQ ID NO:146). In one embodiment, the nucleic acid sequence encoding TRAV19 CDR3 comprises GCTACTTTTCCTAACTTTGGAAATGAGAAATTAACC (SEQ ID NO:147).

[0327] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:72. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 148).

[0328] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 73. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 149).

[0329] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β 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 the TRBV10-3 CDR3 comprises GCCATCAGTGAGTCGGAGCGGTACTACGAGCAGTAC (SEQ ID NO: 152).

[0330] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 153).

[0331] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO:78.In one embodiment, the nucleic acid sequence encoding the TCRβ chain comprises ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCCAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTTCTGTGCCATCAGTGAGTCGGAGCGGTACTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTGA (SEQ ID NO: 154).

[0332] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0333]

[0334] A nucleic acid molecule encoding TCR864 In an embodiment, 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 an embodiment, 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 TRAV17 CDR2 comprises GGATACAAGACAAAA (SEQ ID NO:158). In one embodiment, the nucleic acid sequence encoding TRAV19 CDR3 comprises CTCGTGGTGACTTCAACTCAAATTCCGGGTATGCACTCAAC (SEQ ID NO:159).

[0335] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR alpha chain comprising a constant region comprising the amino acid sequence of SEQ ID NO:84. In one embodiment, the nucleic acid sequence encoding the constant region comprises ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATCACAGACAAAACTGTGTTGGACATGCGCAGCATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC (SEQ ID NO: 160).

[0336] In one embodiment, the nucleic acid molecule encodes a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 85. In one embodiment, the nucleic acid sequence encoding the TCR alpha chain comprises (SEQ ID NO: 161).

[0337] In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β chain comprising one or more of TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3. In an embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR β 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 TTCCCAAGGCACAGTGCA (SEQ ID NO: 163). In one embodiment, the nucleic acid sequence encoding TRBV7-2 CDR3 comprises GCCAGCAAGGTCTATGGCTACACC (SEQ ID NO: 164).

[0338] In one embodiment, the isolated nucleic acid molecule encodes a TCR comprising a TCR beta chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 89. In one embodiment, the nucleic acid sequence encoding the constant region comprises (SEQ ID NO: 165).

[0339] In one embodiment, the nucleic acid molecule encodes a TCR β chain comprising the amino acid sequence of SEQ ID NO:90.In one embodiment, the nucleic acid sequence encoding the TCRβ chain comprises ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCATATCACACAGGAGCTGGAGTCTCCCAGTCCCCCAGTAACAAGGTCACAGAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCATACTGCCCTTTACTGGTACCGACAGAGGCTGGGGCAGGGCCTGGAGTTTTTAATTTACTTCCAAGGCAACAGTGCACCAGACAAATCAGGGCTGCCCAGTGATCGCTTCTCTGCAGAGAGGACTGGGGAATCCGTCTCCACTCTGACGATCCAGCGCACACAGCAGGAGGACTCGGCCGTGTATCTCTGTGCCAGCAAGGTCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGATGAGTGGACACAAGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA (SEQ ID NO: 166).

[0340] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a TCR alpha chain and a TCR beta chain. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a linker domain between the TCR alpha chain and the TCR beta 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).

[0341]

[0342] In certain embodiments, the nucleic acid sequences encoding the alpha and beta chain constant regions of the TCR comprise nucleic acid sequences that are resistant to gene editing, such as CRISPR-mediated gene editing.

[0343] Additionally, the present invention encompasses isolated nucleic acids encoding amino acid sequences having substantial identity to the amino acid sequences disclosed herein, hi certain embodiments, the isolated nucleic acid sequences encode amino acid sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences disclosed herein.

[0344] Additionally, the present invention encompasses isolated nucleic acids having substantial identity to the nucleic acid sequences disclosed herein. In certain embodiments, the isolated nucleic acid sequences have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequences disclosed herein.

[0345] An isolated nucleic acid sequence encoding a polypeptide of the invention can be obtained using any of a number of recombinant methods known in the art, such as, for example, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the same, or by direct isolation from cells or tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0346] The isolated nucleic acid may 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 encoding a polypeptide of the invention, or a functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding a polypeptide of the invention, or a functional fragment thereof.

[0347] The nucleic acid molecules of the present invention can be modified to improve stability in serum or growth medium for cell culture. Modifications can be made to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the present invention and minimize their immunostimulatory properties. For example, to enhance stability, the 3' residues can be stabilized against degradation, e.g., they can be selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0348] In one embodiment of the invention, the nucleic acid molecule may comprise at least one modified nucleotide analogue. For example, the termini may be stabilized by incorporation of modified nucleotide analogues.

[0349] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., modifications to the phosphate sugar backbone). For example, the phosphodiester bond of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides, the phosphoester group connecting adjacent ribonucleotides is replaced by a modified group, for example, a phosphothioate group. In preferred sugar-modified ribonucleotides, the 2'OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, where R is C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.

[0350] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine, are preferred. It should be noted that the above modifications can be combined.

[0351] In some examples, 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 molecule of the present invention may have enhanced resistance to nucleases. To increase nuclease resistance, the nucleic acid molecule can comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. To increase nuclease resistance, the nucleic acid molecule of the present invention can comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, such as 2-amino-A, 2-thio (e.g., 2-thioU), and G-clamp modifications, can also increase binding affinity to targets.

[0352] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0353] The invention also includes vectors into which the isolated nucleic acids of the invention are inserted. The art is replete with suitable vectors that are useful in the present invention.

[0354] Briefly, expression of the natural or synthetic nucleic acid encoding the peptide of the present invention is typically achieved by operably linking the nucleic acid encoding the peptide or a part thereof to a promoter and incorporating the construct into an expression vector.The vector used is suitable for replication and optional integration in eukaryotic cells.Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0355] The vector of the present invention 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. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entirety of which is incorporated herein by reference. In another embodiment, the present invention provides a gene therapy vector.

[0356] The isolated nucleic acids of the present invention can be cloned into several 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.

[0357] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0358] Many virus-based systems have been developed for gene transfer 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 into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of interest either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0359] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors are superior to vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added 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 become powerful gene delivery tools for the treatment of a variety of disorders. AAV vectors possess many features that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0360] In certain embodiments, the vector also contains conventional control elements operably linked to the transgene in a manner that allows its transcription, translation and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences acting 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 (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, if necessary, sequences that promote secretion of the encoded product. Numerous expression control sequences, including promoters that are natural, constitutive, inducible, and / or tissue-specific, are known in the art and can be utilized.

[0361] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but recently, many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, spacing between promoter elements can be as much as 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0362] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0363] Enhancer sequences found on vectors also regulate the expression of genes contained therein. Enhancers usually bind protein factors to enhance the transcription of genes. Enhancers may be located upstream or downstream of the gene they regulate. Enhancers may also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present invention contain one or more enhancers to promote the transcription of genes present in the vector.

[0364] To assess the expression of the peptide, the expression vector introduced into the cell can also contain either a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells that are desired to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection method. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0365] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present or expressed in the recipient organism or tissue and encode a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted 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 obtained commercially. Generally, the construct with the smallest 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be connected to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0366] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0367] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle gun, microinjection, electroporation, etc. 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.

[0368] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors may 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.

[0369] 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 for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0370] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo) is contemplated. In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or combined with micelles, or otherwise associated with lipids. The lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to a particular structure in solution. For example, they can exist as micelles or in bilayer structures with a "collapsed" structure. They may also simply be dispersed in a solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring lipids or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, aminoalcohols, aldehydes, and their derivatives.

[0371] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO. Dicetyl phosphate ("DCP") can be obtained from K&K Laboratories, Plainview, NY. Cholesterol ("Choi") can be obtained from Calbiochem-Behring. Dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc., Birmingham, AL. Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components rearrange themselves before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have a different structure in solution than the normal vesicle structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0372] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as, for example, Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as, for example, detecting the presence or absence of a particular peptide by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0373] In one embodiment, the isolated nucleic acid encoding the polypeptide of the present invention comprises in vitro transcribed (IVT) RNA. The RNA is generated by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA.

[0374] In one embodiment, the DNA used in the PCR comprises an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest, a portion of the gene. The gene may include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene may include exons and introns. In one embodiment, the DNA used in the PCR is a human gene. In another embodiment, the DNA used in the PCR is a human gene including the 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that includes portions of a gene that are linked together to form an open reading frame that encodes a fusion protein. The portions of DNA that are linked together may be from a single organism or from multiple organisms.

[0375] Genes that can be used as a source of DNA for PCR include genes that code for polypeptides that provide a therapeutic or preventative effect to an organism, or genes that can be used to diagnose a disease or disorder in the organism. Preferred genes are those that are useful for short-term treatment, or where there are safety concerns regarding the dosage or the expressed gene. For example, for the treatment of cancer, autoimmune disease, parasitic, viral, bacterial, fungal or other infections, the expressed transgene may code for a polypeptide that functions as a ligand or receptor for cells of the immune system, or can function to stimulate or inhibit the immune system of the organism. In some embodiments, it is not desirable to extend the continued stimulation of the immune system, nor is it necessary to create changes that continue after successful treatment, as this may induce new problems. In the treatment of autoimmune disease, it may be desirable to inhibit or suppress the immune system during a flare-up, but not long-term, as this may make the patient overly susceptible to infections.

[0376] PCR is used to generate templates for in vitro transcription of mRNA used for transfection; methods for performing PCR are well known in the art. Primers used in PCR are designed to have a region that is substantially complementary to a region of DNA used as a template for PCR. As used herein, "substantially complementary" refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence is capable of annealing or hybridizing with a DNA target of interest under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of a DNA template. For example, primers can be designed to amplify a portion of a gene that is normally transcribed in a cell (open reading frame), including the 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 a human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to position 5 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 are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' of the DNA sequence to be amplified relative to the coding strand.

[0377] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from a number of sources.

[0378] Chemical structures with the ability to promote stability and / or translation efficiency can also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between 0 and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be altered by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0379] The 5' and 3' UTRs may be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest may help to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence may reduce the stability of mRNA. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0380] In one embodiment, the 5'UTR may contain the Kozak sequence of the endogenous gene. Alternatively, if a 5'UTR that is not endogenous to the gene of interest has been added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but it appears that it is not necessary for all RNAs to allow efficient translation. The requirement of the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5'UTR may 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.

[0381] To be able to synthesize RNA from a DNA template without the need for gene cloning, a transcription promoter must be added to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase 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 one 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 T7, T3 and SP6 promoters are known in the art.

[0382] In a preferred embodiment, the mRNA has a cap at both the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and stability mRNA in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase generates long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3'UTR results in a normal-sized mRNA, which is ineffective for eukaryotic transfection even after transcription.

[0383] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of a 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)).

[0384] The traditional method to incorporate polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability. Therefore, plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions or other abnormalities. This makes the cloning procedure not only tedious and time-consuming but also often unreliable. Therefore, a method that can construct DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0385] The polyA / T segment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (size 50-5000T). Or it can be generated after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail provides stability to the RNA and reduces RNA 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 between 100 and 5000 adenosines.

[0386] The poly(A) tail of the RNA can be further extended after in vitro transcription using a poly(A) polymerase such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to between 300-400 nucleotides increases the translation efficiency of the RNA by approximately 2-fold. Furthermore, attachment of different chemical groups to the 3' end can increase the stability of the mRNA. Such attachments may include modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. The ATP analogs can further increase the stability of the RNA.

[0387] 5' caps on also provide stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (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)).

[0388] The RNA generated by the method disclosed herein may also include an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes initiation of translation. Any solute suitable for cell electroporation may be included, which may include factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0389] RNA can be introduced into target cells using any of several different methods, including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts) or GenePulserII (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0390] In another embodiment, RNA constructs can be delivered to cells by electroporation.See, for example, US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1 for the formulation and methodology of electroporation of nucleic acid constructs into mammalian cells.Various parameters, including electric field strength, required for electroporation of any known cell type are generally known in the relevant research literature, as well as many patents and patent applications in the field.See, for example, US Patent No. 6,678,556, US Patent No. 7,171,264, and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, for example, MedPulse™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, CA) and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation can also be used to transfect cells in vitro, for example, as described in US20070128708A1. Electroporation can also be utilized to deliver nucleic acids to cells in vitro. Thus, administration of nucleic acids, including expression constructs, to cells via electroporation, utilizing any of the many available devices and electroporation systems known to those skilled in the art, presents an exciting new means to deliver RNA of interest to target cells.

[0391] Engineered Cells In certain embodiments, the compositions of the invention include cells modified to contain or express the peptides of the invention. 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.

[0392] In some embodiments, nucleic acid sequence is delivered to cells using retroviral or lentiviral vectors.For example, retroviral and lentiviral vectors expressing the peptides of the present invention can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transduced cells as carriers, or using cell-free local or systemic delivery of encapsulated, bound or naked vectors.The method used can be used for any purpose where stable expression is necessary or sufficient.

[0393] In other embodiments, the nucleic acid sequence is delivered to cells using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered to various types of eukaryotic cells, as well as tissues and whole organisms, using transfected cells as carriers, or using cell-free local or systemic delivery of encapsulated, conjugated, or naked vectors. The method used can be used for any purpose where transient expression is necessary or sufficient.

[0394] In certain embodiments, the cell may be any suitable cell type capable of expressing the desired peptide. In certain embodiments, the modified cell is used in a method in which the cell is introduced into a recipient. In certain embodiments, the cell is autologous, allogeneic, syngeneic or xenogeneic with respect to the recipient. In certain embodiments, the cell is derived from a stem cell or progenitor cell. In some embodiments, the stem cell or progenitor cell from which the modified cell is derived is autologous, allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0395] In one embodiment, the cell is an immune cell. For example, in certain embodiments, the composition comprises an immune cell comprising or expressing 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 γδ 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, and 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 γδ T cells), B cells, antigen-presenting cells (APCs), natural killer (NK) cells, and NK T cells.

[0396] In one embodiment, the cell is an antigen-presenting cell (APC).For example, in certain embodiments, the composition comprises an APC modified to include or express the mRAS peptide described herein.Exemplary APCs include, but are not limited to, dendritic cells (DCs), macrophages, Langerhans cells, B cells, etc.

[0397] The disclosed compositions and methods can be applied to modulating T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infectious diseases, and autoimmune diseases, including evaluating the ability of genetically modified T cells to kill targeted cancer cells.

[0398] Prior to the expansion and genetic modification of the T cells of the present invention, a source of T cells is obtained from a subject. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any of a variety of techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution may lack calcium and may lack magnesium, or may lack many, if not all, divalent cations. Again, surprisingly, the initial activation step in the absence of calcium leads to expanded activation. As one of skill in the art will readily appreciate, the wash step can be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells are washed by, for example, washing the cells with a centrifuge containing Ca. 2+ Does not contain Mg 2+ The cells can be resuspended in a variety of biocompatible buffers, such as PBS-free, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in media.

[0399] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a Percoll™ gradient or by counterflow centrifugal elutriation. + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads, such as DYNABEADS® M-450CD3 / CD28T, for a time sufficient to positively select the T cells of interest. In one embodiment, the time is about 30 minutes. In a further embodiment, the time ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In one preferred embodiment, the incubation time is 24 hours. When isolating T cells from leukemia patients, longer incubation times, such as 24 hours, can be used to increase the yield of cells. In situations where there are fewer T cells compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or immune-compromised individuals, longer incubation times can be used to separate T cells. Additionally, longer incubation times can be used to increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time that T cells are allowed to bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially or adversely selected at the beginning of culture or at other times during the process. Additionally, subpopulations of T cells can be preferentially or adversely selected at the beginning of culture or at other desired times by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

[0400] Enrichment of a T cell population by negative selection can be achieved with a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, the monoclonal antibody cocktail typically includes antibodies against CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich or positively select for regulatory T cells expressing T. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0401] For isolation of the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may 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 a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used. Using a higher concentration can increase cell yield, cell activation, and cell proliferation. Additionally, using a higher cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or cells from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, using a higher concentration of cells can increase the number of CD8 T cells that normally express weakly CD28. + T cells can be selected more efficiently.

[0402] In a related embodiment, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and the cells can be minimized. This allows for the selection of cells that express large amounts of the desired antigen that binds to the particles. For example, CD4 + T cells express high levels of CD28 and sparse concentrations of CD8 + In one embodiment, the concentration of cells used is 5×10 6In another embodiment, the concentration used is about 1×10 5 / ml~1×10 6 / ml, and any integer value therebetween.

[0403] In other embodiments, cells can be incubated on a rotator at various speeds for various lengths of time, either at 2-10 °C or at room temperature.

[0404] The T cells for stimulation can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more homogenous product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this regard, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1° per minute and storing them in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing at -20°C or liquid nitrogen.

[0405] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest at room temperature for 1 hour before being activated using the methods of the invention.

[0406] Also contemplated in the context of the present invention is the collection of a blood sample or apheresis product from a subject at a time period before the expanded cells described herein are needed. The source of cells to be expanded can be collected at any time needed, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, the blood sample or apheresis is taken from a generally healthy subject. In a particular embodiment, the blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease, but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In a particular embodiment, the T cells are expanded, frozen, and can be used later. In a particular embodiment, the sample is collected from the patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant therapies, including but not limited to treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, FK506, antibodies, or other immunosuppressants such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These agents either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (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 a further embodiment, the cells are isolated for a patient and frozen for later use in combination with (e.g., before, simultaneously with, or after) T cell ablative therapy using either bone marrow or stem cell transplant, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or Campath. In another embodiment, the cells can be isolated prior to B cell ablative therapy, such as an agent reactive with CD20, e.g., Rituxan, and frozen for later use in therapy.

[0407] In a further embodiment of the invention, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved for their ability to expand ex vivo immediately after treatment during the period when the patient is normally recovering from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, in the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a state of the subject in which repopulation, recirculation, regeneration, and / or expansion of certain cell types is particularly favored, particularly during a defined time frame after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0408] Whether before or after genetic modification of the T cells to express the peptides of the invention, the T cells can be activated and expanded generally using methods described, for example, in U.S. Pat. Nos. 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.

[0409] In general, the T cells of the present invention are expanded by contact with a surface having attached thereto an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, the T cell population can be stimulated as described herein, such as by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Co-stimulation of accessory molecules on the surface of T cells uses a ligand that binds to the accessory molecule. For example, the T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. CD4 + T cells or CD8 +To stimulate the proliferation of T cells, anti-CD3 antibody and anti-CD28 antibody. Examples of anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), and can be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0410] In certain embodiments, the primary and costimulatory signals of the T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or bound to a surface. If bound to a surface, the agents may be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent may be bound to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that binds to the agent. In this regard, see, for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present invention.

[0411] 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 an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same bead with comparable molecular weights. In one embodiment, the CD4 + A 1:1 ratio of each antibody bound to beads for T cell proliferation and T cell growth is used. In a particular embodiment of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell proliferation is observed compared to the proliferation observed using a 1:1 ratio. In one particular embodiment, an increase of about 1-fold to about 3-fold is observed compared to the proliferation observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one embodiment of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than 1. In a particular embodiment of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to beads is greater than 2:1. In one particular embodiment, a CD3:CD28 ratio of 1:100 of antibodies bound to beads is used. In another embodiment, a CD3:CD28 ratio of 1:75 of antibodies bound to beads is used. In a further embodiment, a CD3:CD28 ratio of 1:50 of antibody bound to beads is used. In another embodiment, a CD3:CD28 ratio of 1:30 of antibody bound to beads is used. In one preferred embodiment, a CD3:CD28 ratio of 1:10 of antibody bound to beads is used. In another embodiment, a CD3:CD28 ratio of 1:3 of antibody bound to beads is used. In yet another embodiment, a CD3:CD28 ratio of 3:1 of antibody bound to beads is used.

[0412] Particle to cell ratios ranging from 1:500 to 500:1, and any integer values ​​therebetween, can be used to stimulate T cells or other target cells. As one of ordinary skill in the art can readily appreciate, the ratio of particles to cells can depend on the particle size relative to the target cells. For example, small sized beads can only bind a small number of cells, while larger beads can bind many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1, and any integer values ​​therebetween, and in further embodiments, the ratio includes 1:9 to 9:1, and any integer values ​​therebetween, and can be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells resulting in T cell stimulation can vary as described above, but 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 one preferred ratio being at least 1:1 particle per T cell. In one embodiment, a particle to cell ratio of 1:1 or less is used. In one particular embodiment, the preferred particle:cell ratio is 1:5. In a further embodiment, the particle to cell ratio can be varied depending on the day of stimulation. For example, in one embodiment, the particle to cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day for up to 10 days to a final ratio of 1:1 to 1:10 (based on the cell number on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to 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 adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day of stimulation and 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that a variety of other ratios may be suitable for use in the present invention.In particular, the ratio varies with particle size and cell size and type.

[0413] In a further embodiment of the present invention, cells such as T cells are combined with drug-coated beads, the beads and cells are subsequently separated, and then the cells are cultured.In an alternative embodiment, before culturing, the drug-coated beads and cells are not separated, but are cultured together.In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, to increase the ligation of cell surface markers, thereby inducing cell stimulation.

[0414] By way of example, cell surface proteins can be linked by contacting the T cells with paramagnetic beads (3x28 beads) having anti-CD3 and anti-CD28 attached thereto. In one embodiment, the cells (e.g., 10 4 From 10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (without divalent cations such as calcium or magnesium). Again, one skilled in the art can readily appreciate that any cell concentration can be used. For example, the target cells may be very rare in the sample and may consist of only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of the target cells of interest. Thus, any cell number is within the context of the present invention. In certain embodiments, it may 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, more than 100 million cells / ml is used. In further embodiments, a concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml can be used. Using a high concentration can increase cell yield, cell activation, and cell proliferation. Additionally, using a high cell concentration allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and, in certain embodiments, would be desirable to obtain. For example, using a high concentration of cells can increase the cell yield, cell activation, and cell proliferation of CD8 T cells that normally have weak expression of CD28. + T cells can be selected more efficiently.

[0415] In one embodiment of the invention, the mixture can be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of time in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, 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. Several cycles of stimulation may also be desirable, so that the culture time of the T cells is 60 days or more. Suitable conditions for T cell culture include an appropriate medium (e.g., basal medium or RPMI medium 1640, or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for the growth of cells known to one of skill in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The medium may contain RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in an amount sufficient for the growth and proliferation of T cells. Antibiotics such as penicillin and streptomycin are included only in the experimental cultures, not in the culture of cells injected into the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0416] T cells exposed to different stimulation times may exhibit different properties. For example, a typical blood or peripheral blood mononuclear cell product obtained by apheresis may have either cytotoxic or suppressor T cell populations (T C , CD8 + ) than the helper T cell population (T H , CD4 +Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors occurs primarily in T cells before about day 8–9. H After about 8-9 days, the population of T cells becomes increasingly more numerous. C Therefore, depending on the purpose of treatment, mainly T H It may be advantageous to inject a population of T cells containing T C If an antigen-specific subset of cells has been isolated, it may be beneficial to further expand this subset.

[0417] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but for the most part, are reproducible over the course of the cell expansion process. Such reproducibility thus allows the ability to tailor activated T cell products for specific purposes.

[0418] method The present invention provides a method for treating a subject having or suspected of having mRAS-associated cancer. The method can be used to treat any cancer associated with a mutation in RAS, such as a mutation at G12, including hematological malignancies, solid tumors, primary or metastatic tumors.

[0419] Exemplary tumors and cancer types treatable by the present invention include 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical SCC, ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (ESCC), and esophageal squamous cell carcinoma (ESC). SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumors (NETs).

[0420] In certain embodiments, the present invention provides a method for inducing immune response against mRAS in a subject.For example, administering the composition described herein is used to induce specific immune response, including T cell-mediated immune response against mRAS and cancer cells expressing mRAS.In certain examples, inducing immune response against mRAS leads to inhibition of tumor growth and tumor cell death.

[0421] In one embodiment, the method comprises contacting the subject with a composition of the invention. 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 contain or express an mRAS peptide described herein. In certain embodiments, the method comprises contacting the subject with a composition comprising a polypeptide comprising a TCR described herein, a nucleic acid molecule encoding a polypeptide comprising a TCR described herein, or a cell modified to express a TCR described herein.

[0422] In certain embodiments, the subject is identified as having the HLA type associated with the mRAS peptide that TCR binds.For example, as described herein, in certain instances, TCR binds to a specific mRAS peptide in the context of a specific HLA molecule.Thus, in certain embodiments, the method comprises identifying the subject as having a specific HLA molecule, and then administering to the subject the composition that comprises or codes for TCR as described herein. For example, in one embodiment, a method includes identifying a subject as having an HLA-A*11:01 molecule and administering to the subject a composition comprising a TCR, a nucleic acid encoding a TCR, or a cell expressing a TCR, where 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, a method includes identifying a subject as having a particular HLA molecule, and then administering to the subject a composition comprising or encoding a particular mRAS peptide as described herein. For example, in one embodiment, a method includes identifying a subject as having an HLA-A*11:01 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 includes 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).

[0423] In one embodiment, the method includes 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 includes 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 includes 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).

[0424] In one embodiment, the method includes 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, where 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 includes identifying a subject as having an HLA-A*02:01 molecule and administering to the subject a composition comprising a mRAS peptide, a nucleic acid encoding a mRAS peptide, or a cell expressing an mRAS peptide, where 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).

[0425] In one embodiment, the method includes 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, where the TCR specifically binds to an 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 includes 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 an mRAS peptide, where 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).

[0426] In one embodiment, the method includes identifying the subject as having a specific RAS mutation. For example, in one embodiment, the method includes identifying the subject as having a specific mutation relative to G12 of wild-type RAS. For example, in one embodiment, the method includes identifying the subject as having a G12C, G12D, G12R, or G12V mutation compared to wild-type RAS.

[0427] In one embodiment, the method includes 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 is KLVVVGA C In one embodiment, the method includes identifying a subject as having an HLA-A*02:01 allele and a G12C RAS ​​mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide is KLVVVGA. C GV (SEQ ID NO: 1).

[0428] In one embodiment, the method includes 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 is KLVVVGA D In one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide is KLVVVGA. D GV (SEQ ID NO:2).

[0429] In one embodiment, the method includes 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 is KLVVVGA R In one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide is KLVVVGA. R GV (SEQ ID NO:3).

[0430] In one embodiment, the method includes 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 is V In one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide is KLVVVGA. V GV (SEQ ID NO: 4).

[0431] In one embodiment, the method includes 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 is C GVGK (SEQ ID NO: 5), or VVVGA CIn one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising VVGA (SEQ ID NO: 6). C GVGK (SEQ ID NO: 5), or VVVGA C Contains GVGK (sequence number: 6).

[0432] In one embodiment, the method includes 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 is D GVGK (SEQ ID NO: 7), or VVVGA D In one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising VVGA (SEQ ID NO: 8). D GVGK (SEQ ID NO: 7), or VVVGA D Contains GVGK (sequence number: 8).

[0433] In one embodiment, the method includes 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 is R GVGK (SEQ ID NO: 9), or VVVGA RIn one embodiment, the method includes identifying a subject as having an HLA-A*03:01 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 is VVGA R GVGK (SEQ ID NO: 9), or VVVGA R Contains GVGK (sequence number: 10).

[0434] In one embodiment, the method includes 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 is V GVGK (SEQ ID NO: 11), or VVVGA V In one embodiment, the method includes identifying a subject as having an HLA-A*03:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising GVGK (SEQ ID NO: 12). V GVGK (SEQ ID NO: 11), or VVVGA V Contains GVGK (sequence number: 12).

[0435] In one embodiment, the method includes 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 is C GVGK (SEQ ID NO: 5), or VVVGA CIn one embodiment, the method includes identifying a subject as having an HLA-A*11:01 allele and a G12C RAS ​​mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising VVGA (SEQ ID NO: 6). C GVGK (SEQ ID NO: 5), or VVVGA C Contains GVGK (sequence number: 6).

[0436] In one embodiment, the method includes 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 is D GVGK (SEQ ID NO: 7), or VVVGA D In one embodiment, the method includes 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 the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising GVGK (SEQ ID NO: 8). D GVGK (SEQ ID NO: 7), or VVVGA D Contains GVGK (sequence number: 8).

[0437] In one embodiment, the method includes 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 is R GVGK (SEQ ID NO: 9), or VVVGA RIn one embodiment, the method includes identifying a subject as having an HLA-A*11:01 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 is VVGA R GVGK (SEQ ID NO: 9), or VVVGA R Contains GVGK (sequence number: 10).

[0438] In one embodiment, the method includes identifying a subject as having an HLA-A*11:01 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 is V GVGK (SEQ ID NO: 11), or VVVGA V In one embodiment, the method includes identifying a subject as having an HLA-A*11:01 allele and a G12V RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide is VVGA V GVGK (SEQ ID NO: 11), or VVVGA V Contains GVGK (sequence number: 12).

[0439] In one embodiment, the method includes identifying a subject as having an HLA-B*07:02 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 is CIn one embodiment, the method includes identifying a subject as having an HLA-B*07:02 allele and a G12C RAS ​​mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising GAVGKSAL (SEQ ID NO: 13). C Contains GVGKSAL (SEQ ID NO: 13).

[0440] In one embodiment, the method includes identifying a subject as having an HLA-B*07:02 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 is D In one embodiment, the method includes identifying a subject as having an HLA-B*07:02 allele and a G12D RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 14). D Contains GVGKSAL (SEQ ID NO: 14).

[0441] In one embodiment, the method includes identifying a subject as having an HLA-B*07:02 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 is R In one embodiment, the method includes identifying a subject as having an HLA-B*07:02 allele and a G12R RAS mutation, and administering to the subject a composition comprising an mRAS peptide, a nucleic acid encoding the mRAS peptide, or a cell expressing the mRAS peptide, wherein the mRAS peptide specifically binds to an mRAS peptide comprising GVGKSAL (SEQ ID NO: 15). R Contains GVGKSAL (sequence number: 15).

[0442] Subjects can be identified as being of a particular HLA type or having a particular RAS mutation using any method known in the art, including but not limited to DNA sequencing, RNA sequencing, next generation sequencing, PCR, immunoassays, etc.

[0443] In one embodiment, the method comprises administering at least one cell genetically modified to express TCR, wherein TCR specifically binds to RAS, mRAS, or fragments thereof.For example, in certain embodiments, the method comprises administering a cell genetically modified to express TCR, wherein TCR specifically binds to mRAS peptide with mutation corresponding to G12.In certain embodiments, the method comprises administering a cell genetically modified to express TCR, wherein TCR specifically binds to mRAS peptide with mutation G12C, G12D, G12R, or G12V at the position corresponding to G12.

[0444] In one embodiment, the invention includes cell therapy in which cells are modified to contain or express an mRAS peptide or TCR of the invention and the cells are infused into a recipient in need thereof.

[0445] In one embodiment, the invention includes a cell therapy, wherein the method comprises administering to a subject a composition comprising cells, such as antigen presenting cells, that contain or express an mRAS peptide as described herein. For example, in one embodiment, the method comprises administering to a subject a composition comprising antigen presenting cells that are loaded with an mRAS peptide as described herein and express the mRAS peptide on their surface.

[0446] In one embodiment, the invention includes a cell therapy, the method comprising administering to a subject a composition comprising cells activated or stimulated by antigen presenting cells comprising or expressing an mRAS peptide as described herein. For example, in one embodiment, the method comprises contacting cells, such as naive T cells, with antigen presenting cells that are loaded with an mRAS peptide as described herein and express the mRAS peptide on their surface, thereby activating the cells. The method comprises administering to a subject a composition comprising the activated cells. For example, in one embodiment, the method of the invention comprises the steps of: (1) providing a population of naive T cells; (2) providing a population of dendritic cells; (3) loading or pulsing recipient cells with one or more mRAS peptides as described herein; (4) co-culturing the naive T cells with the loaded dendritic cells; and (4) isolating the stimulated T cells. In one embodiment, the method further comprises the step (5) of administering the stimulated T cells to a subject in need thereof, for example a subject having, suspected of having, or at risk of having an mRAS-associated cancer.

[0447] In certain embodiments, the injected cells (e.g., antigen-presenting cells that present an mRAS peptide) can stimulate an immune response in vivo. For example, in certain embodiments, the injected cells can activate or stimulate endogenous immune cells to target and kill tumor cells in the recipient.

[0448] In certain embodiments, the injected cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, in certain instances, the modified cells are capable of replicating in vivo, resulting in long-term persistence and surveillance that may lead to sustained tumor control.

[0449] In one embodiment, the modified T cells of the invention can undergo strong in vivo T cell expansion and persist for an extended period of time. In another embodiment, the modified T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth. For example, the modified T cells of the invention can undergo strong in vivo T cell expansion and persist at high levels in the blood and bone marrow for an extended period of time to form specific memory T cells.

[0450] The compositions of the present invention can be administered as pharmaceutical compositions alone or in combination with diluents and / or other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can be combined with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients to constitute the compositions described herein. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, etc.; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0451] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the condition of the patient, the type and severity of the patient's disease, and the appropriate dosage may be determined by clinical trials.

[0452] When an "immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0453] Administration of the subject compositions (e.g., compositions comprising a TCR, a nucleic acid molecule encoding a TCR, or genetically modified cells expressing a TCR) can be by any convenient method, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneally. In one embodiment, the compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions of the invention are administered by intravenous injection. In a particular embodiment, the compositions are injected directly into a tumor or lymph node.

[0454] In one embodiment, the invention provides a method of treating cancer comprising treating a subject with a complementary cancer therapy, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormonal therapy, or a combination thereof, prior to, concurrently with, or following administration of a composition of the invention.

[0455] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, sodium), altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, recombinant interferon alpha-2a, paclitaxel, teniposide, and streptozotocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, diam- Drogalactitol, fluorodopan, hepsulfan, hycanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), mitotic inhibitors. (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mithramycin, mitomycin,Daunorubicin, VP-16-213, VM-26, navelbine, and taxotere), biologics (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridines, bisanthophylls, HCl, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyldaunomycin, oxantrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0456] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene and roloxifene). Examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0457] In further embodiments, the compositions of the invention are administered to the patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using any of the chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the compositions of the invention are administered after B cell ablative therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0458] In certain embodiments, the compositions of the invention are administered during surgical removal or debulking of a tumor or diseased tissue. For example, in a subject undergoing surgical treatment of a diseased tissue or tumor, the composition can be administered to the site to further treat the tumor.

[0459] Subjects to which administration of the compositions and pharmaceutical compositions of the invention is contemplated include, but are not limited to, mammals, including humans and other primates, non-human primates, commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs.

[0460] The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-accepted practices. Strategies for administration and scheduling of T cells have been discussed (Ertl et al, 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[0461] kit The present invention also includes a kit comprising a composition comprising an mRAS peptide of the present invention, a nucleic acid molecule encoding the mRAS peptide, a cell comprising or expressing the mRAS peptide, a polypeptide comprising a TCR, a nucleic acid molecule encoding a TCR, a cell expressing a TCR, or a combination thereof, and instructional materials that describe the use of the composition. For example, in some embodiments, the instructional materials describe administering the composition or a combination thereof to an individual as a therapeutic treatment or a non-therapeutic use, as described elsewhere herein. In one embodiment, the kit further comprises a pharma- ceutically acceptable carrier (optionally sterile) suitable for dissolving or suspending the therapeutic composition of the present invention, e.g., prior to administering the composition to an individual. Optionally, the kit comprises an applicator for administering the composition. In certain embodiments, the kit comprises a reagent used to identify the HLA type of the subject. In certain embodiments, the kit comprises a reagent used to identify a RAS mutation (e.g., a mutation at position G12) in the subject. EXAMPLES

[0462] Experimental Example The present invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the present invention should in no way be construed as being limited to these examples, but rather as encompassing all variations that become evident as a result of the teachings provided herein.

[0463] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following examples are not to be construed as limiting the remainder of the disclosure.

[0464] Example 1: Identification of mRAS neoantigens As described herein, various computational and proteomic studies have been performed to identify mRAS neoantigens and their interactions with HLA types. Figure 1 is a schematic showing the mutant RAS epitope discovery strategy, where in silico models are used to predict the affinity of mRAS peptides to MHC, followed by experiments to measure the affinity / stability of the interaction and detect the peptides by mass spectrometry.

[0465] An in silico study was performed to predict mRAS neoantigens utilizing antigen.garnish software, which analyzes human or mouse DNA missense mutations, insertions, deletions, and fusions and computationally predicts neoepitopes using seven validated algorithms. The model outputs neoepitopes by MHC I / II binding affinity. For example, as shown in Figure 2, the model was used to predict a 9-10 mer neoepitope containing a mutation at the position corresponding to G12 of RAS.

[0466] As shown in Figure 3 and Figure 4A and Figure 4B, the model predicted the binding of mRAS neoantigens to different HLA class I alleles. The table in Figure 4B summarizes the mRAS short peptides predicted to bind to specific HLA types.

[0467] Experiments were also performed to examine peptide-MHC binding. A fluorescence polarization assay using competitive binding of peptides of interest is shown in FIG. 5. This assay was used to measure the affinity of various mRAS peptide sequences for the HLA class I alleles of interest outlined in the table in FIG. 5. Additional data showing peptide binding in a fluorescence polarization assay is shown in FIG. 6A. Experiments were also performed to study peptide stability by scintillation proximity assay. The designations for the mutant RAS peptides shown in FIG....

Claims

1. An immunogenic composition comprising a mutant RAS peptide that contains a mutation relative to G12 of wild-type RAS.

2. The composition of claim 1 , wherein the peptide comprises a G12C, G12D, G12R, or G12V mutation.

3. The composition of claim 1 , wherein the mutant RAS peptide comprises 9 or 10 amino acid residues.

4. The composition of claim 1, wherein the mutant RAS peptide comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from SEQ ID NOs: 1-16.

5. The composition of claim 1, wherein the mutant RAS peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-16.

6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a mutant RAS peptide which comprises a mutation relative to G12 of wild-type RAS.

7. A cell that contains or has been engineered to express a mutant RAS peptide that contains a mutation relative to G12 of wild-type RAS.

8. The cell of claim 7 , wherein the cell is an immune cell.

9. The cell of claim 10, wherein 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, and a NK T cell.

10. A method of inducing an immune response in a subject, comprising administering to the subject the immunological composition of claim 1.

11. The method of claim 10, wherein the method comprises identifying the HLA type of a subject and administering to the subject a composition comprising or encoding a mutant RAS peptide comprising a mutation at a relative position to G12 of wild-type RAS, wherein the mutant RAS peptide binds to the identified HLA molecule of the subject.

12. The method of claim 10, wherein the subject has or is at risk of having a RAS-associated cancer.

13. The cancers include 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical squamous cell carcinoma (CLL), and uterine cancer.

13. The method of claim 12, wherein the cancer is selected from the group consisting of: ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (esophageal SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

14. The method of inducing an immune response in a subject includes: a. contacting a cell with a composition comprising a mutant RAS peptide that contains a mutation relative to G12 of wild-type RAS, thereby stimulating the cell; and b. administering the stimulated cells to the subject.

15. 15. The method of claim 14, wherein the method comprises contacting naive T cells of the subject with an antigen-presenting cell that presents a mutant RAS peptide, thereby stimulating the T cells.

16. The method of claim 14, wherein the cells are autologous to the subject.

17. The method of claim 15, wherein the T cells and antigen presenting cells are autologous to the subject.

18. A composition comprising 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.

19. 20. The composition of claim 18, wherein the RAS peptide comprises a mutation at a position corresponding to G12 compared to wild-type RAS.

20. 20. The composition of claim 19, wherein the mutation in the mRAS peptide corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V, compared to wild-type RAS.

21. 20. The composition of claim 18, wherein 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.

22. 20. The composition of claim 18, wherein the TCR comprises TRAV39 CDR1, TRAV39 CDR2, TRAV39 CDR3, TRBV20-1 CDR1, TRBV20-1 CDR2, and TRBV20-1 CDR3.

23. 20. The composition of claim 18, wherein 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.

24. The composition of claim 18, wherein the TCR comprises TRAV12-1 CDR1, TRAV12-1 CDR2, TRAV12-1 CDR3, TRBV28 CDR1, TRBV28 CDR2, and TRBV28 CDR3.

25. 20. The composition of claim 18, wherein 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.

26. The composition of claim 18, wherein the TCR comprises TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV10-3 CDR1, TRBV10-3 CDR2, and TRBV10-3 CDR3.

27. 20. The composition of claim 18, wherein 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.

28. The composition of claim 18, wherein the TCR comprises TRAV17 CDR1, TRAV17 CDR2, TRAV17 CDR3, TRBV11-2 CDR1, TRBV11-2 CDR2, and TRBV11-2 CDR3.

29. 20. The composition of claim 18, wherein 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.

30. 20. The composition of claim 18, wherein the TCR comprises TRAV19 CDR1, TRAV19 CDR2, TRAV19 CDR3, TRBV9 CDR1, TRBV9 CDR2, and TRBV9 CDR3.

31. 20. The composition of claim 18, wherein 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.

32. The composition of claim 18, wherein the TCR comprises TRAV4 CDR1, TRAV4 CDR2, TRAV4 CDR3, TRBV7-2 CDR1, TRBV7-2 CDR2, and TRBV7-2 CDR3.

33. The composition of claim 18, wherein the composition comprises a fusion polypeptide comprising a TCR alpha chain and a TCR beta chain.

34. The composition of claim 33 , wherein the fusion polypeptide comprises a linker domain.

35. 35. The composition of claim 34, wherein the linker domain is a cleavable linker domain.

36. A composition comprising an isolated nucleic acid molecule encoding the composition of any one of claims 18 to 36.

37. 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.

38. The cell of claim 37, wherein the mRAS peptide contains a mutation at a position corresponding to G12 compared to wild-type RAS.

39. 39. The cell of claim 38, wherein the mutation in the mRAS peptide corresponds to a mutation selected from the group consisting of G12C, G12D, G12R, and G12V compared to wild-type RAS.

40. The cell of claim 37 , wherein the cell is modified to express a fusion polypeptide comprising a TCR alpha chain and a TCR beta chain.

41. The cell of claim 37, wherein the cell is genetically modified by introduction of an isolated nucleic acid molecule encoding a polypeptide comprising at least one of a TCR alpha chain and a TCR beta chain.

42. The cell of claim 37 , wherein the cell is an immune cell.

43. 43. The cell of claim 42, wherein the immune cell is selected from the group consisting of a T cell, a NK cell, and a NK T cell.

44. The cell of claim 37, wherein the cell is autologous to a subject with a RAS-associated cancer.

45. 38. The cells of claim 37, wherein the cells are autologous to the 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.

46. A method of treating a subject having a cancer associated with mRAS, comprising administering to the subject a cell according to any one of claims 37 to 45.

47. The subject is a patient with 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 squamous cell carcinoma (lung SCC), small cell lung cancer (SCLC), renal papillary cancer, hepatocellular carcinoma (HCC), breast cancer, cervical squamous cell carcinoma (CLL), and / or uterine cancer.

47. The method of claim 46, wherein the patient has a cancer selected from the group consisting of: ovarian adenocarcinoma, adrenal carcinoma, prostate cancer, neuroblastoma, glioblastoma multiforme (GBM), medulloblastoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma (esophageal SCC), osteosarcoma, sarcoma, and small intestinal neuroendocrine tumor (NET).

48. 47. The method of claim 46, wherein the method comprises identifying the HLA type of the subject.

49. 47. The method of claim 46, wherein the method comprises isolating one or more cells of the subject and modifying the one or more cells to express the TCR.

50. The method of claim 46, wherein the method comprises modifying the one or more cells to express the TCR by contacting the one or more cells with an isolated nucleic acid molecule encoding one or more of a TCR alpha chain and a TCR beta chain.

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